JPH03167198A - Inhibitor of angiotensin-transforming material - Google Patents
Inhibitor of angiotensin-transforming materialInfo
- Publication number
- JPH03167198A JPH03167198A JP1303294A JP30329489A JPH03167198A JP H03167198 A JPH03167198 A JP H03167198A JP 1303294 A JP1303294 A JP 1303294A JP 30329489 A JP30329489 A JP 30329489A JP H03167198 A JPH03167198 A JP H03167198A
- Authority
- JP
- Japan
- Prior art keywords
- angiotensin
- converting enzyme
- fraction
- solution
- inhibition rate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000000463 material Substances 0.000 title abstract 2
- 239000003112 inhibitor Substances 0.000 title 1
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 24
- 150000003839 salts Chemical class 0.000 claims abstract description 6
- 239000005541 ACE inhibitor Substances 0.000 claims description 9
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 claims description 8
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 8
- 101710129690 Angiotensin-converting enzyme inhibitor Proteins 0.000 claims description 5
- 101710086378 Bradykinin-potentiating and C-type natriuretic peptides Proteins 0.000 claims description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 abstract description 11
- 108010073771 Soybean Proteins Proteins 0.000 abstract description 11
- 235000013305 food Nutrition 0.000 abstract description 11
- 235000019710 soybean protein Nutrition 0.000 abstract description 10
- 235000010469 Glycine max Nutrition 0.000 abstract description 6
- 244000068988 Glycine max Species 0.000 abstract description 6
- 238000004587 chromatography analysis Methods 0.000 abstract description 6
- 239000002808 molecular sieve Substances 0.000 abstract description 6
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 abstract description 6
- 102000057297 Pepsin A Human genes 0.000 abstract description 5
- 108090000284 Pepsin A Proteins 0.000 abstract description 5
- 229940111202 pepsin Drugs 0.000 abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 3
- 229940124572 antihypotensive agent Drugs 0.000 abstract 1
- 239000003795 chemical substances by application Substances 0.000 abstract 1
- 239000005526 vasoconstrictor agent Substances 0.000 abstract 1
- 102100030988 Angiotensin-converting enzyme Human genes 0.000 description 44
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 description 41
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 41
- 230000005764 inhibitory process Effects 0.000 description 32
- 239000000243 solution Substances 0.000 description 24
- 230000000694 effects Effects 0.000 description 15
- 108010064733 Angiotensins Proteins 0.000 description 14
- 102000015427 Angiotensins Human genes 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 238000002835 absorbance Methods 0.000 description 10
- 230000002401 inhibitory effect Effects 0.000 description 10
- 238000000034 method Methods 0.000 description 9
- 230000031700 light absorption Effects 0.000 description 8
- 238000010521 absorption reaction Methods 0.000 description 7
- 230000036772 blood pressure Effects 0.000 description 7
- 102400000344 Angiotensin-1 Human genes 0.000 description 6
- 101800000734 Angiotensin-1 Proteins 0.000 description 6
- ORWYRWWVDCYOMK-HBZPZAIKSA-N angiotensin I Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 ORWYRWWVDCYOMK-HBZPZAIKSA-N 0.000 description 6
- 238000005194 fractionation Methods 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 239000008057 potassium phosphate buffer Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N ethyl acetate Substances CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- NHJVRSWLHSJWIN-UHFFFAOYSA-N 2,4,6-trinitrobenzenesulfonic acid Chemical compound OS(=O)(=O)C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O NHJVRSWLHSJWIN-UHFFFAOYSA-N 0.000 description 3
- WFIYPADYPQQLNN-UHFFFAOYSA-N 2-[2-(4-bromopyrazol-1-yl)ethyl]isoindole-1,3-dione Chemical compound C1=C(Br)C=NN1CCN1C(=O)C2=CC=CC=C2C1=O WFIYPADYPQQLNN-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 3
- 238000005341 cation exchange Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- -1 foods Natural products 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- FZERHIULMFGESH-UHFFFAOYSA-N N-phenylacetamide Chemical compound CC(=O)NC1=CC=CC=C1 FZERHIULMFGESH-UHFFFAOYSA-N 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- 235000010724 Wisteria floribunda Nutrition 0.000 description 2
- 239000005018 casein Substances 0.000 description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
- 235000021240 caseins Nutrition 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 229930014626 natural product Natural products 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- FAKRSMQSSFJEIM-MLWJPKLSSA-N (2s)-1-(2-methyl-3-sulfanylpropanoyl)pyrrolidine-2-carboxylic acid Chemical compound SCC(C)C(=O)N1CCC[C@H]1C(O)=O FAKRSMQSSFJEIM-MLWJPKLSSA-N 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- JXNRXNCCROJZFB-RYUDHWBXSA-N Tyr-Arg Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 JXNRXNCCROJZFB-RYUDHWBXSA-N 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- 229960001413 acetanilide Drugs 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 108010016268 hippuryl-histidyl-leucine Proteins 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 229910000043 hydrogen iodide Inorganic materials 0.000 description 1
- 108010053037 kyotorphin Proteins 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 239000000820 nonprescription drug Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 229940001941 soy protein Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野］
この発明は、アンギオテンシンＩをアンギオテンシン■
に変換させるアンギオテンシン変換酵素の活性を阻害す
るアンギオテンシン変換酵素阻害物質に関するものであ
る．
〔従来技術］
従来より、アンギオテンシン変換酵素がアンギオテンシ
ン■をアンギオテンシン■に変換させる作用を有するこ
とが知られている．そして、このようにアンギオテンシ
ン変換酵素によってアンギオテンシンＩから変換された
アンギオテンシン■が血圧を上昇させる作用を有すると
言われている．
このため、アンギオテンシンＩをアンギオテンシン■に
変換させるアンギオテンシン変換酵素の活性を阻害すれ
ば、血圧の上昇を抑制することができるとして、従来よ
り、上記のようなアンギオテンシン変換酵素の括性を阻
害する物質について様々な研究が行われ、種々のアンギ
オテンシン変換酵素阻害物質が開発されるに至った．
そして、上記のようなアンギオテンシン変換酵素の活性
を阻害する物質として、従来においては、例えば、Ｄ−
２−メチル−３−メルカプトプロパノイルーし−プロリ
ンのような合成物質の他、特開昭６２−２７０５３３号
公報，特開昭６４−５４９７号公報，特開昭６４−８３
０９６号公報等においてカゼインを分解させて得られる
各種のベプチドが開示されている．しかし、上記の合成
物がらなるアンギオテ；シン変換酵素阻’！ｌ質は、一
般にアンギオテ：シン変換酵素の阻害活性が高いが、合
戒物で２るため、多くの場合、副作用等の安全性の点１
若干問題を有していた．
一方、カゼイン等を分解して得られるアンｊオテンシン
変換酵素阻害物質の場合、上記の４成物からなるものに
比べ、その阻害活性が一Ｒに低いが、その原料が天然物
、特に食品由来σものであるため、一般にその毒性が低
く、安３性の高いものであった．
このため、上記のように天然物、特に食品ｄ来のアンギ
オテンシン変換酵素阻害物質にっｂて、さらなる開発が
望まれていた．
［発明が解決しようとする課題］
この発明は、上記のような事情に鑑みなされたものであ
り、アンギオテンシンＩをアンギオテンシン■に変換さ
せるアンギンテンシン変捕酵素の活性を有効に阻害して
、血圧の上昇を抑制できると共に、人体に対する安全性
も高く、食品等として投与され、マイルドな作用で血圧
の上昇を抑制することができるアンギオテンシン変換酵
素阻害物質を提供することを課題とするものである．
［課題を解決するための手段］
この発明においては、アンギオテンシン■をアンギオテ
ンシンＨに変換させるアンギオテンシン変換酵素の活性
を阻害するアンギオテンシン変換酵素阻害物質として、
下記に示す構造式［１１，［２］のべブチド及びその塩
の少なくとも一種を用いるようにしたのである．［ １
３ Ａｓｐ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ−Ａｒｇ−
Ｖａｌ−Ｐｈｅ［ ２ ］ Ｔｙｒ−Ａｒｇ−１ｌ
ｅ−Ｌｅｕ−Ｇｌｕ−Ｐｈｅここで、上記ベプチドにお
ける塩の形態としては、塩酸塩，臭化水素酸塩，ヨウ化
水素塩，硫酸塩，燐酸塩等の無機酸塩、酢酸塩，トリフ
ルオロ酢酸塩，クエン酸塩，マレイン酸塩，フマル酸塩
，酒石酸塩，乳酸塩，メタンスルホン酸塩，Ｐ一トルエ
ンスルホン酸塩等の有機酸塩が挙げられる．
また、上記ペプチドを構成するアミノ酸は、天然に存在
するという点で、Ｌ一体であることが望ましい．
ここで、上記［１］．［２］の構造式に示される各ベプ
チドは、大豆から分離された分離大豆蛋白をペプシンに
よって分解させて製造することができる．
そして、上記楕遣式［１］．［２］に示されるペプチド
及びその塩は、アンギオテンシンＩをアンギオテンシン
Ｈに変換させるアンギオテンシン変換酵素の活性を阻害
する作用を有すると共に、上記のように食品として使用
されている大豆から得られた分離大豆蛋白を、ベプシン
によって分解させて製造できる食品由来のものであり、
安全性が高いものとなっている．［実施例］
以下、この発明の実施例について具体的に説明する．
まず、上記［１］，［２］に示す構造式のべプチドを製
造する場合について説明する．この実施例においては、
大豆から分離させた分離大豆蛋白（不二製油■製 フジ
プローＲ〉３．０ｇを水に溶解させ、これに酢酸を加え
てｐＨ３．２に調製し、さらに６規定の塩酸を加えてｐ
Ｈ２．０に調整した溶液６０−とした。[Detailed Description of the Invention] [Industrial Application Field] This invention provides angiotensin I to angiotensin ■
This relates to angiotensin-converting enzyme inhibitors that inhibit the activity of angiotensin-converting enzyme, which converts angiotensin to [Prior Art] It has been known that angiotensin converting enzyme has the effect of converting angiotensin ■ into angiotensin ■. It is said that angiotensin ■, which is converted from angiotensin I by angiotensin converting enzyme, has the effect of increasing blood pressure. For this reason, the increase in blood pressure can be suppressed by inhibiting the activity of angiotensin converting enzyme, which converts angiotensin I to angiotensin Various studies have been conducted and various angiotensin-converting enzyme inhibitors have been developed. Conventionally, as a substance that inhibits the activity of angiotensin converting enzyme as described above, for example, D-
In addition to synthetic substances such as 2-methyl-3-mercaptopropanoyl-proline, JP-A-62-270533, JP-A-64-5497, JP-A-64-83
Various peptides obtained by decomposing casein are disclosed in Publication No. 096 and the like. However, the above-mentioned compounds can inhibit angio-converting enzymes! Generally, lactic acid has a high inhibitory activity of angiote:sin converting enzyme, but since it is a non-prescription drug, in many cases there are safety concerns such as side effects.
There were some problems. On the other hand, in the case of anj-otensin-converting enzyme inhibitors obtained by decomposing casein, etc., the inhibitory activity is only 1R lower than that of those consisting of the four components mentioned above, but the inhibitory activity is only 1R, but the inhibitory activity is only 1R when the raw material is derived from natural products, especially foods. Since it is a σ compound, its toxicity is generally low and its safety is high. Therefore, as mentioned above, further development of angiotensin-converting enzyme inhibitors derived from natural products, particularly foods, has been desired. [Problems to be Solved by the Invention] This invention was made in view of the above circumstances, and effectively inhibits the activity of angintensin morphase, which converts angiotensin I to angiotensin ■, thereby lowering blood pressure. The object of the present invention is to provide an angiotensin-converting enzyme inhibitor that can suppress the increase in blood pressure, is highly safe for the human body, can be administered as food, etc., and can suppress the increase in blood pressure with a mild action. [Means for Solving the Problem] In this invention, as an angiotensin converting enzyme inhibitor that inhibits the activity of angiotensin converting enzyme that converts angiotensin ■ to angiotensin H,
At least one of the bebutides and salts thereof having the structural formulas [11 and 2] shown below is used. [ 1
3 Asp-Gln-Thr-Pro-Arg-
Val-Phe[2]Tyr-Arg-1l
e-Leu-Glu-Phe Here, the salt form of the above peptide includes inorganic acid salts such as hydrochloride, hydrobromide, hydrogen iodide, sulfate, and phosphate, acetate, and trifluoroacetic acid. Examples include organic acid salts such as salt, citrate, maleate, fumarate, tartrate, lactate, methanesulfonate, and P-toluenesulfonate. Furthermore, the amino acids constituting the above-mentioned peptide are preferably L-units because they occur naturally. Here, the above [1]. Each peptide shown in the structural formula [2] can be produced by decomposing isolated soybean protein separated from soybeans with pepsin. And the above elliptical formula [1]. The peptide and its salt shown in [2] have the effect of inhibiting the activity of angiotensin converting enzyme that converts angiotensin I to angiotensin H, and as mentioned above, the peptide isolated from soybeans obtained from soybeans used as food. It is a food-derived product that can be produced by breaking down protein with vepsin.
It is highly safe. [Examples] Examples of the present invention will be specifically described below. First, the case of producing peptides having the structural formulas shown in [1] and [2] above will be explained. In this example,
3.0 g of isolated soybean protein (Fuji Pro R manufactured by Fuji Oil Co., Ltd.) separated from soybeans was dissolved in water, acetic acid was added to adjust the pH to 3.2, and 6N hydrochloric acid was further added to pH 3.0.
A solution 60- was adjusted to H2.0.
そして、この溶液にベプシンを上記分離大見蛋白の１／
５００の量、すなわち６ｍｇ加え、これを３７℃で１０
時間反応させ、上記の分離大豆蛋白をベプシンによって
酵素分解させた。Then, add vepsin to this solution and add 1/1/2 of the above separated Omi protein.
500, i.e. 6 mg, and this was heated at 37°C for 10
After a time reaction, the isolated soybean protein was enzymatically degraded by vepsin.
その後、これを１００℃で１５分間加熱し、上記ペプシ
ンを失活させた後、これを遠心分離機により、１０００
０ｒｐｍで２分間遠心分離させて、沈殿画分と溶液画分
とに分離させ、沈殿画分を除去し、溶液画分だけを取り
出した．次いで、このようにして取り出された溶液画分
を、分子ふるいクロマトグラフィ−（ファルマシア社製
セファデックスＧ−２５）を用いて分画するようにし
た．
ここで、上記の分子ふるいクロマトグラフィーによって
分画を行うにあたっては、カラム容積が２００ｄのもの
を使用し、また展開液としては０．０５Ｍの酢酸溶液を
使用し、流量が４ｍ／ｈｒとなるようにして、各フラク
ションに３−づつ分取するようにした。Thereafter, this was heated at 100°C for 15 minutes to inactivate the pepsin, and then centrifuged at 1000°C.
The mixture was centrifuged at 0 rpm for 2 minutes to separate into a precipitate fraction and a solution fraction, the precipitate fraction was removed, and only the solution fraction was taken out. Next, the solution fraction thus taken out was fractionated using molecular sieve chromatography (Sephadex G-25, manufactured by Pharmacia). Here, when performing fractionation by the above-mentioned molecular sieve chromatography, a column with a volume of 200 d was used, a 0.05 M acetic acid solution was used as the developing solution, and the flow rate was adjusted to 4 m/hr. Then, 3 fractions were collected in each fraction.
次いで、このようにして分取された各フラクションのも
のについて、それぞれ蛋白量及びアンギオテンシン変換
酵素阻害率を測定するようにした．
ここで、蛋白量を測定するにあたり、この実施例におい
ては、公知のＴＮＢＳ法を用いて測定するようにした。Next, the protein content and angiotensin converting enzyme inhibition rate of each fraction thus collected were measured. In this example, the protein amount was measured using a known TNBS method.
即ち、この実施例においては、０．１０％のＴＮＢＳ
（２，４．６−トリニトロベンゼンスルホン酸〉溶液を
０．５−と、Ｏ．ＯＩＭの亜硫酸ナトリウム溶液を０．
５ｄと、０．１５Ｍのホウ酸緩衝液を２．０一加えたも
のに、上記の各フラクションにおけるサンプルを吸光度
の測定範囲に入るように適当に希釈したものを０．５一
加えて、これらを３７℃で６０分間反応させた後、分光
光度計によって４２０ｎｍの光の吸収を測定し、その結
果を、第１図に実線で示した。なお、上記分光光度計に
よって測定された４２０ｎｍの光の吸収が大きいほど、
サンプル中におけるベプチド数が多くなっている．
また、上記のように分取された各フラクションにおける
アンギオテンシン変換酵素阻害率を測定するにあたり、
この実施例においては、公知のアンギオテンシン変換酵
素阻害活性測定法（Ｃｕｓｈｍａｎ−Ｃｈｅｕｎｇ法）
によってアンギオテンシン変換酵素阻害率を測定するよ
うにした．
ここで、この実施例においては、ラビットラングアセト
ンパウダー（シグマ社製）１０ｇを、１００ｎｄの５０
ｍＭホウ酸［１液（ｐＨ８．３〉に溶解させ、４０００
０ｇ，４０分の遠心処理を行った後、その上清液を５０
ｍＭのリン酸カリウム緩衝液で５倍に希釈して、アンギ
オテンシン変換酵素溶液を調製した．一方、上記の各フ
ラクションのものを凍結乾燥させた後、これらのものを
それぞれ５０ｍＭのリン酸カリウム緩衝液０．６−に溶
解させ、この溶液よりサンプルとしてそれぞれ５０μｊ
採取した．
そして、このように採取した５０μｇの各サンプルに対
して、それぞれ基質Ｂｚ−Ｇｌｙ−Ｈｉｓ−Ｌｅｕ（ペ
ブチド研究所製〉と、リン酸カリウム緩衝液と塩化ナト
リウムとの混合溶液と、上記のように調製したアンギオ
テンシン変換酵素溶液５０μ１とを加えて２５０μｊに
した．この時、上記溶液中における基質の最終濃度は２
．５ｍＭ、リン酸カリウム緩衝液の最終濃度は１００ｍ
Ｍ、塩化ナトリウムの最終濃度は３００ｍＭであった．
次いで、このように調製された各混合溶液を３７℃で３
０分間反応させた後、１規定の塩酸を２５０μｇ添加さ
せて反応を停止させ、その後、１．５−の酢酸エチルを
加えて１０秒間攪拌し、さらに３５０００ｒｐｍで２分
間遠心分離させて、酢酸エチル層を１一採取した．そし
て、このように採取されたものを、エバボレートによっ
て乾固させた後、これを１−の蒸留水に溶解させ、抽出
されたヒブリル酸の吸収（２２８ｎｍの吸光度〉を測定
し、各フラクションにおけるサンプルのアンギオテンシ
ン変換酵素阻害率を、下記の［１］式によって求めるよ
うにした．
（但し、ＯＤ．は上記のようにしてサンプルを加えて測
定した時の吸光度，ＯＤ−ｂは上記の混合溶液を反応さ
せる前に１規定の塩酸を２５０μｌ添加させて測定した
時の吸光度，ＯＤ，は上記の混合溶液にサンプルを加え
ずに測定した時の吸光度．ＯＤｃｂは上記の混合溶液に
サンプルを加えずに１規定の塩酸を２５０μ｛添加させ
て測定した時の吸光度．）
そして、このようにして測定した各フラクＳヨンのサン
プルにおけるアンギオテンシン変毛酵素阻害率（ＡＣＥ
阻害率）を第１図に破線Ｉ示した。That is, in this example, 0.10% TNBS
(2,4.6-trinitrobenzenesulfonic acid) solution at 0.5- and O.OIM sodium sulfite solution at 0.5-.
5d and 2.0 parts of 0.15M borate buffer, and 0.5 parts of the samples in each of the above fractions diluted appropriately so as to fall within the absorbance measurement range. After reacting at 37° C. for 60 minutes, the absorption of light at 420 nm was measured using a spectrophotometer, and the results are shown in FIG. 1 as a solid line. Note that the greater the absorption of 420 nm light measured by the spectrophotometer,
The number of peptides in the sample is large. In addition, in measuring the angiotensin converting enzyme inhibition rate in each fraction collected as described above,
In this example, a known method for measuring angiotensin-converting enzyme inhibitory activity (Cushman-Cheung method) was used.
The angiotensin-converting enzyme inhibition rate was measured using the following method. Here, in this example, 10 g of Rabbit Lang Acetone Powder (manufactured by Sigma) was
Dissolved in mM boric acid [1 solution (pH 8.3), 4000
After centrifuging at 0g for 40 minutes, the supernatant was
An angiotensin converting enzyme solution was prepared by diluting it 5 times with mM potassium phosphate buffer. On the other hand, after freeze-drying each of the above fractions, each of these fractions was dissolved in 0.6-mM potassium phosphate buffer, and 50 μj of each sample was prepared from this solution.
Collected. Then, 50 μg of each sample collected in this way was treated with the substrate Bz-Gly-His-Leu (manufactured by Pebutide Institute), a mixed solution of potassium phosphate buffer, and sodium chloride, and as described above. 50 μl of the prepared angiotensin converting enzyme solution was added to make 250 μj. At this time, the final concentration of the substrate in the above solution was 2.
．． 5mM, final concentration of potassium phosphate buffer is 100mM
M, the final concentration of sodium chloride was 300mM. Next, each mixed solution prepared in this way was incubated at 37°C for 3
After reacting for 0 minutes, 250 μg of 1 N hydrochloric acid was added to stop the reaction, then 1.5-ethyl acetate was added and stirred for 10 seconds, and further centrifuged at 35,000 rpm for 2 minutes to dissolve ethyl acetate. One layer was sampled. After drying the sample in this manner with evaborate, it was dissolved in 1-distilled water, and the absorption (absorbance at 228 nm) of the extracted hypohybrilic acid was measured. The angiotensin converting enzyme inhibition rate was calculated using the following formula [1]. (However, OD. is the absorbance measured by adding the sample as described above, and OD-b is the absorbance measured by adding the sample as described above. The absorbance when measured by adding 250 μl of 1N hydrochloric acid before the reaction, OD, is the absorbance when measured without adding a sample to the above mixed solution. ODcb is the absorbance when measured without adding a sample to the above mixed solution. 250μ of 1N hydrochloric acid (absorbance when measured).) Then, the angiotensin hair enzyme inhibition rate (ACE) of each fraction sample measured in this way was
Inhibition rate) is shown in FIG. 1 by a broken line I.
この結果、同図に示すように、アンギオテ〉シン変換酵
素阻害率は、そのフラクション番角が４４〜６０のもの
において大きくなってした．
このため、この実施例においては、フラクション番号が
４４〜６０の部分、すなわち分子ｌが約２０００〜２０
０のものを分取するようにした．
そして、このようにして分取されたものを神結乾燥させ
、次いで、このように凍結乾燥されたものを、分子ふる
いクロマトグラフィー（ファルマシア社製 セファデッ
クスＧ−１０）を使用してさらに分画するようにした．
ここで、この分子ふるいクロマトグラフィーによって分
画を行うにあたっては、カラム容積が１５０−になった
ものを使用し、また展開液としでは０．０５Ｍの酢酸溶
液を使用し、流量が３ｍＲ／ｈｒとなるようにして、各
フラクションに１１１ｔｆＩづつ分取するようにした．
次いで、このようにして各フラクションに分取されたも
のについて、上記の場合と同様にして、各フラクション
における蛋白量及びアンギオテンシン変換酵素阻害率を
測定するようにした．
そして、分光光度計によって測定された各フラクション
における４２０ｎｍの光の吸収結果を第２図に実線で示
す一方、各フラクションがら採取された各サンプルにお
けるアンギオテンシン変換酵素阻害率（ＡＣＥ阻害率）
を同図に破線で示した．
この結果、第２図に示すように、フラクション番号が１
０５〜１３０のものにおいては、ベプチド数に対するア
ンギオテンシン変換酵素阻害率が高くなっていた．
このため、この実施例においては、フラクション番号が
１０５〜１３０のもの、すなわち分子量が約ｉｏｏｏ〜
４００になったものを分取するようにした．
そして、このようにして分取したものを、今度は陽イオ
ン交換分離によって、分画するようにした．
ここで、陽イオン交換分離を行うにあたっては、ファル
マシア社製のＦＰＬＣユニットを使用すると共に、カラ
ムとして陽イオン交換樹脂Ｍｏｎｏ Ｓ ＨＲ・５
／５（商品名）を使用し、また展開液として、０．０５
Ｍの酢酸一アンモニアＭＩ衝液（ｐＨ７．５）を用いる
一方、グラジエントとして、０．５Ｍの塩化ナトリウム
溶液を用い、この塩化ナトリウム溶液を加える量を当初
から５分間はＯにし、５〜２５分の間でＯ〜１００％に
増加させるようにし、流量がｌＷＩｉ／−となるように
して、各フラクションに０．５−づつ分取するようにし
た．
そして、上記のようにして分画された各フラクションの
ものについて、それぞれ蛋白量及びアンギオテンシン変
換酵素阻害率を測定するようにした．
ここで、各フラクシ３ンにおけるアンギオテンシン変換
酵素阻害率を測定するにあたっては、各フラクションの
ものを凍結乾燥させた後、これらのものをそれぞれ５０
ｍＭのリン酸カリウム緩衝液１ｒＩｒ１に溶解させるよ
うにし、それ以外については、上記のアンギオテンシン
変換酵素阻害活性測定法と同様にして、各フラクション
におけるサンプルについてそれぞれアンギオテンシン変
換酵素阻害率（ＡＣＥ阻害率）を測定し、その結果を第
３図（Ａ）に示した．一方、各フラクションにおけるサ
ンプルについての蛋白量の測定は、公知の紫外部吸光法
によって行い、２８０ｎｍの光の吸収を測定し、その結
果を、第３図（Ｂ）に示した．なお、この場合において
も、２８０ｎｍの光の吸収が大きいほど、サンプル中に
おける蛋白量が多くなっている．
この結果、上記の第３図（Ａ）に示したように、０．５
Ｍの塩化ナトリウム溶液を加えてぃない時点における素
通り画分においては、アンギオテンシン変換酵素阻害率
が低くなっていた．
そして、この実施例のものにおいては、第３図（Ａ）に
おいて、アンギオテンシン変換酵素阻害率が高くなった
フラクション番号が２８．２９のものと、フラクション
番号が３８．３９のものとの２つのピークのものを分取
するようにした．
次いで、このようにして分取したフラクション番号が２
８．２９のものと、フラクション番号が３８．３９のも
のとをそれぞれ凍結乾燥させ、これらの２つのサンプル
をそれぞれ４０μｇの蒸留水に溶解させた後、それぞれ
高速液体クロマトグラフィ−（■日立製作所製のＨＰＬ
Ｃ Ｌ−４２００）を用いて、さらに分画を行った．
ここで、上記高速液体クロマトグラフィーによる分画に
おいては、０．０５％のテトラヒドロフルフリルアクリ
レート（ＴＦＡ）と５％のアセトアニリドとを加えたＡ
溶液と゛、０．０５％のＴＦＡと１００％のアセトアニ
リドとを加えたＢ溶液とを用い、当初から５分間は上記
Ａ溶液だけを加え、５〜３５分の間で、上記Ａ溶液を１
００〜Ｏ％に減少させる一方、Ｂ溶液をＯ〜１００％に
増加させるようにして分画を行った．
そして、このように分画されたものについて、前記の紫
外部吸光法により、その蛋白量を測定するようにした．
この結果、フラクション番号が２８．２９のものをサン
プルとして用いたものにおいては、２８０ｎｍの光に対
して、第４図に示すような光の吸収結果が得られた．
そして、同図において光の吸収がある程度有り、適当な
量め蛋白を含んでいる（ａ）の部分を分取し、このよう
に分取された（ａ）の部分のものを凍結乾燥させた後、
これを０．４−の蒸留水に溶解させ、この溶液を２０μ
Ｊ採取したものに、５０ｍＭのリン酸カリウム緩衝液３
０μｇを加えて５０μ１になったサンプルを調製し、前
記のアンギオテンシン変換酵素阻害活性測定法と同様に
して、アンギオテンシン変換酵素阻害率を測定したとこ
ろ、このサンプルにおけるアンギオテンシン変換酵素阻
害率は３９．９％と高い値を示した．
また、フラクション番号３８．３９のものをサンプルと
して用いたものにおいては、２８０ｎｍの光に対して、
第５図に示すような光の吸収結果が得られた．
そして、同図において光の吸収が高く、蛋白量が多くな
っている（ｂ）の部分について、上記（ａ）の部分の場
合と同様にして、アンギオテンシン変換酵素阻害率を測
定したところ、この（ｂ）の部分におけるサンプルにお
いても、アンギオテンシン変換酵素阻害率が３７．４％
と高い値を示した．
次いで、上記の第４図に示す（ａ）の部分のものと、第
５図に示す（ｂ）の部分のものとをそれぞれ分取し、こ
れらのものをそれぞれ凍結乾燥させて、完全に溶剤を除
去した後、上記の場合と同様にして、再度、上記の高速
液体クロマトグラフィーによって精製を行い、その蛋白
量を前記の紫外部吸光法によって測定するようにした．
この結果、第４図に示す（ａ）の部分から精製されたも
のにおいては、２８０ｎｍの光に対して、第６図に示す
ような３つの山になった光の吸収結果が得られた．
そして、同図において、第１，第２，第３の冬山部分（
ａｘ ）．（ａ２），（ａ３）について、それぞれ前記
のようにしてアンギオテンシン変換酵素阻宵率を測定し
たところ、（ａ１〉の部分はアンギオテンシン変換酵素
阻害率がほぼＯ％であり、（ａ２）の部分はアンギオテ
ンシン変換酵素阻害率が３３．１％と高い値を示し、（
ａ，）の部分はアンギオテンシン変換酵素阻害率が１６
．１％であった．
このため、（ａ）の部分からは、上記のようにアンギオ
テンシン変換酵素阻害率が高くなった（ａ２）の部分を
分取するようにした．一方、第５図に示す（ｂ）の部分
がら精製之れたちのにおいては、２８０ｎｍの光に対し
て、第７図に示すような１つの山になった光グ吸収結果
が得られた。As a result, as shown in the figure, the angiothecin converting enzyme inhibition rate was greater in the fraction number 44-60. Therefore, in this example, the portion with fraction numbers 44 to 60, that is, the molecule l is about 2000 to 20
The sample with a value of 0 was collected. The fraction thus collected is dried, and then the freeze-dried product is further fractionated using molecular sieve chromatography (Sephadex G-10 manufactured by Pharmacia). I decided to do so. Here, when performing fractionation by this molecular sieve chromatography, a column with a volume of 150 - is used, and a 0.05M acetic acid solution is used as a developing solution, with a flow rate of 3 mR/hr. 111tfI was collected in each fraction.
Next, the protein content and angiotensin converting enzyme inhibition rate in each fraction were measured in the same manner as described above for each fraction separated in this way. The absorption results of 420 nm light in each fraction measured by a spectrophotometer are shown in solid lines in Figure 2, while the angiotensin converting enzyme inhibition rate (ACE inhibition rate) in each sample collected from each fraction is shown in Figure 2.
is shown by a broken line in the same figure. As a result, as shown in Figure 2, the fraction number is 1.
In those with numbers 05 to 130, the angiotensin converting enzyme inhibition rate relative to the number of peptides was high. Therefore, in this example, the fraction number is 105 to 130, that is, the molecular weight is about iooo to
I decided to sort out those that reached 400. The fraction collected in this way was then fractionated using cation exchange separation. Here, in performing cation exchange separation, we used an FPLC unit manufactured by Pharmacia, and used a cation exchange resin Mono S HR-5 as a column.
/5 (product name) and as a developing solution, 0.05
A 0.5M sodium chloride solution was used as the gradient, and the amount of sodium chloride added was set to 0 for 5 minutes from the beginning, and for 5 to 25 minutes. The flow rate was set to 1WIi/-, and 0.5- to each fraction was collected. Then, the protein content and angiotensin converting enzyme inhibition rate were measured for each fraction fractionated as described above. Here, to measure the angiotensin converting enzyme inhibition rate of each fraction, after freeze-drying each fraction, 50%
The angiotensin converting enzyme inhibition rate (ACE inhibition rate) was determined for each sample in each fraction in the same manner as the above-mentioned angiotensin converting enzyme inhibitory activity measurement method. The results are shown in Figure 3 (A). On the other hand, the protein content of the samples in each fraction was measured by a known ultraviolet absorption method, and the absorption of light at 280 nm was measured, and the results are shown in Figure 3 (B). In this case as well, the greater the absorption of light at 280 nm, the greater the amount of protein in the sample. As a result, as shown in FIG. 3(A) above, 0.5
In the flow-through fraction at the time when the sodium chloride solution of M was not added, the angiotensin converting enzyme inhibition rate was low. In this example, in FIG. 3(A), there are two peaks, one with the fraction number 28.29 where the angiotensin converting enzyme inhibition rate is high and the other with the fraction number 38.39. I decided to separate out the . Next, the fraction number separated in this way is 2.
The fraction number 8.29 and the fraction number 38.39 were freeze-dried, and each of these two samples was dissolved in 40 μg of distilled water. H.P.L.
Further fractionation was performed using CL-4200). Here, in the above-mentioned fractionation by high performance liquid chromatography, A
Using solution B containing 0.05% TFA and 100% acetanilide, only the above solution A was added for the first 5 minutes, and 1% of the above solution A was added between 5 and 35 minutes.
Fractionation was carried out by increasing the B solution from 0 to 100% while decreasing it from 0 to 0%. The amount of protein thus fractionated was then measured by the ultraviolet absorption method described above. As a result, in the sample using fraction number 28.29, light absorption results as shown in Figure 4 were obtained for 280 nm light. Then, in the same figure, the part (a), which has a certain amount of light absorption and contains an appropriate amount of protein, was separated, and the part (a) thus separated was freeze-dried. rear,
Dissolve this in 0.4-distilled water, and add 20μ of this solution.
J samples were added with 50mM potassium phosphate buffer 3
A sample of 50 μl was prepared by adding 0 μg, and the angiotensin converting enzyme inhibition rate was measured in the same manner as the above-mentioned method for measuring angiotensin converting enzyme inhibitory activity. The angiotensin converting enzyme inhibition rate in this sample was 39.9%. showed a high value. In addition, in the sample using fraction number 38.39, for light of 280 nm,
The light absorption results shown in Figure 5 were obtained. In the same figure, the angiotensin converting enzyme inhibition rate was measured in the same manner as in the case of the part (a) above for the part (b) where light absorption is high and the amount of protein is large. Also in the sample in part b), the angiotensin converting enzyme inhibition rate was 37.4%.
showed a high value. Next, the portion (a) shown in FIG. 4 and the portion (b) shown in FIG. After removing the protein, the protein was purified again by the high performance liquid chromatography described above in the same manner as above, and the amount of protein was measured by the ultraviolet absorption method described above. As a result, in the case of the product purified from the part (a) shown in Fig. 4, absorption results of three peaks of light as shown in Fig. 6 were obtained for 280 nm light. In the same figure, the first, second, and third winter mountain portions (
ax). For (a2) and (a3), the angiotensin converting enzyme inhibition rate was measured as described above, and the angiotensin converting enzyme inhibition rate was approximately 0% for the part (a1>), and for the part (a2). The angiotensin converting enzyme inhibition rate was as high as 33.1%, and (
Part a,) has an angiotensin converting enzyme inhibition rate of 16
．． It was 1%. Therefore, from part (a), part (a2), which had a high angiotensin converting enzyme inhibition rate as described above, was separated. On the other hand, when the part (b) shown in FIG. 5 was purified, a single peak of light absorption as shown in FIG. 7 was obtained for 280 nm light.
そして、同図に山で示される部分（ｂ１）Ｌ：ついて，
前記のようにしてアンギオテンシン鵠換酵素阻害率を測
定したところ、アンギオテ冫シン変換酵素阻害率が２８
、４％と高い値を坏したため、この山の部分を分取する
ようにした．
次いで、上記のようにして分取した第６図に示す（ａ２
）の部分のものと、第７図に示す（ｂ＋ ）の部分のも
のとについて、それぞれアミノ酸シーケンサー（アブラ
イズバイオシステ社製の４７７Ａ型）により、これらに
含まれるベプチドの構造を特定した．
この結果、第６図に示す（ａ２）の部分のものは、下記
の構造式［１］に示されるペプチドであることが、また
第７図に示す（ｂ１）の部分のものは、下記の構造式［
２］に示されるペプチドであることが判明した．
［ １ ］ ＡｓＰ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ−Ａ
ｒｇ−Ｖａｌ−Ｐｈｅ［ ２ ］ Ｔｙｒ−Ａｒｇ
−Ｉｌｅ−Ｌｅｕ−Ｇｌｕ−Ｐｈｅそして、このように
して得られた上記構造式［１１．［２］の各ベブチドに
ついて、前記のアンギオテンシン変換酵素阻害活性測定
法（Ｃｕｓｈｍａｎ−Ｃｈｅｕｎｇ法）によって、アン
ギオテンシン変換酵素阻害率が５０％になるのに必要な
ベプチドの濃度ＩＤ，ｏを測定したところ、上記構造式
［１］に示されるベプチドにおいては、そのＩＤ，．が
４２μＭであり、また上記構造式［２］に示されるベプ
チドにおいては、そのＩＤ，ｏが１５６μＭであった。Then, regarding the part (b1) L shown by the mountain in the same figure,
When the angiotensin converting enzyme inhibition rate was measured as described above, the angiotensin converting enzyme inhibition rate was 28.
Since the value was as high as 4%, we decided to separate this mountain part. Next, as shown in FIG. 6, the sample was fractionated as described above (a2
) and (b+) shown in Figure 7, the structures of the peptides contained therein were identified using an amino acid sequencer (Model 477A manufactured by Arise Biosystems). As a result, the part (a2) shown in Figure 6 is the peptide shown by the following structural formula [1], and the part (b1) shown in Figure 7 is the peptide shown by the following structural formula [1]. Structural formula[
It turned out to be the peptide shown in [2]. [1] AsP-Gln-Thr-Pro-A
rg-Val-Phe[2]Tyr-Arg
-Ile-Leu-Glu-Phe and the above structural formula [11. For each peptide in [2], the concentration ID,o of the peptide required to achieve an angiotensin converting enzyme inhibition rate of 50% was measured by the angiotensin converting enzyme inhibitory activity measurement method (Cushman-Cheung method) described above. In the peptide shown in the above structural formula [1], its ID, . was 42 μM, and the ID,o of the peptide represented by the above structural formula [2] was 156 μM.
また、上記構造式［１］，［２］に示される各ペプチド
が、その原料として使用した分離大豆蛋白に由来するも
のであるかを確認したところ、上記の構造式［１］に示
されるアミノ酸の配列は、大豆蛋白における蛋白質画分
がｌｌｓＡ５Ａ４ Ｂ３サブユニット プリカーサー配
列中に存在しており、また上記の構造式［２］に示され
るアミノ酸の配列は、大豆蛋白における蛋白質画分が７
８α′サブユニット配列中に存在しており、上記構造式
［１］．［２］に示される各ベプチドが、その原料に使
用した分離大豆蛋白に由来するものであることが確認さ
れた．
従って、上記構造式［１Ｆ．［２］に示される各ベプチ
ドは、上記のようにアンギオテンシン■をアンギオテン
シン■に変換させるアンギンテンシン変換酵素に対して
、その活性を有効に阻害することができ、食品等として
投与することによって、血圧の上昇をマイルドな作用で
抑制することが期待でき、またこれらの各ペプチドは、
上記のように食品として利用されている大豆蛋白に由来
するものであるため、副作用等の問題がなく、人体に対
する安全性も高いものであった．
［発明の効果］
以上詳述したように、この発明に係るアンギオテンシン
変換酵素阻害物質は、アンギオテンシンＩをアンギオテ
ンシン■に変換させるアンギオテンシン変換酵素の活性
をマイルドな作用で有効に阻害することができると共に
、食品として使用されている大豆から得られた分離大豆
蛋白を、ペプシンによって分解させて製造できる食品由
来のものであり、副作用等がなく、安全性が高いものと
なっていた．
この結果、この発明に係るアンギオテンシン変換酵素阻
害物質は、人体に対する安全性が高く、食品等として投
与して、マイルドな作用で血圧を下げることができ、ま
た高血圧の予防効果も期待できるものであった．In addition, after confirming whether each peptide shown in the above structural formulas [1] and [2] was derived from the isolated soybean protein used as a raw material, we found that the amino acids shown in the above structural formula [1] The protein fraction in soybean protein exists in the llsA5A4 B3 subunit precursor sequence, and the amino acid sequence shown in the above structural formula [2] indicates that the protein fraction in soybean protein exists in the llsA5A4 B3 subunit precursor sequence.
It exists in the 8α' subunit sequence and has the above structural formula [1]. It was confirmed that each peptide shown in [2] was derived from the isolated soybean protein used as the raw material. Therefore, the above structural formula [1F. Each of the peptides shown in [2] can effectively inhibit the activity of angintensin-converting enzyme, which converts angiotensin ■ to angiotensin ■, as described above, and by administering it as a food etc., blood pressure can be reduced. It is expected that each of these peptides will suppress the increase in
As mentioned above, since it is derived from soy protein, which is used as a food, there were no problems such as side effects, and it was highly safe for the human body. [Effects of the Invention] As detailed above, the angiotensin-converting enzyme inhibitor according to the present invention can effectively inhibit the activity of angiotensin-converting enzyme that converts angiotensin I to angiotensin ■ with a mild action, and It is a food-derived product that can be produced by decomposing isolated soybean protein obtained from soybeans used as food with pepsin, and has no side effects and is highly safe. As a result, the angiotensin-converting enzyme inhibitor according to the present invention is highly safe for the human body, can be administered as food, etc., to lower blood pressure with a mild effect, and can also be expected to have a preventive effect on hypertension. Ta.
図面はいずれもこの発明の実施例を示す図であり、第１
図は分子ふるいクロマトグラフィー（ファルマシア社製
セファデックスＧ−２５〉によって分画された各フラ
クションにおける４２０ｎｍの光に対する吸光度及びア
ンギオテンシン変換酵素阻害率を示す図、第２図は分子
ふるいクロマトグラフイー（ファルマシア社製 セファ
デックスＧ−１０）によって分画された各フラクション
における４２０ｎｍの光に対する吸光度及びアンギオテ
ンシン変換酵素阻害率を示す図、第３図（Ａ），（Ｂ）
は陽イオン交換分離によって分画された各フラクション
におけるアンギオテンシン変換酵素阻害率及び２８０ｎ
ｍの光に対する吸光度を示す図、第４図は高速液体クロ
マトグラフィーを用いて最初マトグラフィーを用いて再
度分画を行った場合における２８０ｎｍの光に対する吸
光度を示す図である．The drawings are all diagrams showing embodiments of the present invention, and the first
The figure shows the absorbance to 420 nm light and angiotensin converting enzyme inhibition rate in each fraction fractionated by molecular sieve chromatography (Pharmacia Sephadex G-25). Figures 3 (A) and (B) show the absorbance to 420 nm light and angiotensin converting enzyme inhibition rate in each fraction fractionated using Sephadex G-10 (manufactured by Sephadex G-10).
is the angiotensin converting enzyme inhibition rate and 280n in each fraction fractionated by cation exchange separation.
FIG. 4 is a diagram showing the absorbance for light at 280 nm when fractionation is first performed again using high performance liquid chromatography.
Claims (1)
びその塩の少なくとも一種を含有することを特徴とする
アンギオテンシン変換酵素阻害物質。 ［１］Ａｓｐ−Ｇｌｎ−Ｔｈｒ−Ｐｒｏ−Ａｒｇ−Ｖａ
ｌ−Ｐｈｅ［２］Ｔｙｒ−Ａｒｇ−Ｉｌｅ−Ｌｅｕ−Ｇ
ｌｕ−Ｐｈｅ[Scope of Claims] 1. An angiotensin converting enzyme inhibitor characterized by containing at least one of the peptides and salts thereof represented by the following structural formulas [1] and [2]. [1] Asp-Gln-Thr-Pro-Arg-Va
l-Phe[2]Tyr-Arg-Ile-Leu-G
lu-Phe
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1303294A JPH0725793B2 (en) | 1989-11-24 | 1989-11-24 | Angiotensin converting enzyme inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1303294A JPH0725793B2 (en) | 1989-11-24 | 1989-11-24 | Angiotensin converting enzyme inhibitor |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6113396A Division JP2535782B2 (en) | 1994-05-02 | 1994-05-02 | Angiotensin converting enzyme inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03167198A true JPH03167198A (en) | 1991-07-19 |
| JPH0725793B2 JPH0725793B2 (en) | 1995-03-22 |
Family
ID=17919222
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1303294A Expired - Lifetime JPH0725793B2 (en) | 1989-11-24 | 1989-11-24 | Angiotensin converting enzyme inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0725793B2 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08269087A (en) * | 1992-03-02 | 1996-10-15 | Fumio Yamauchi | New tetrapeptide and pentapeptide, their production and antihypertensive containing the same as active ingredient |
| US6232438B1 (en) | 1996-01-11 | 2001-05-15 | Nong Shim Co., Ltd. | Angiotensin converting enzyme inhibitors |
| WO2002055546A1 (en) * | 2001-01-16 | 2002-07-18 | Ajinomoto Co.,Inc. | Angiotensin converting enzyme inhibitors |
| WO2004011016A1 (en) * | 2002-07-30 | 2004-02-05 | Tropicana Products, Inc. | Cardiovascular health enhancement with soy fortified orange juice compositions |
| JP2007326790A (en) * | 2006-06-06 | 2007-12-20 | Aomori Prefecture | Method for producing acetic acid-enzyme treated product of apios americana, peptide derived from apios americana and method for producing the same |
-
1989
- 1989-11-24 JP JP1303294A patent/JPH0725793B2/en not_active Expired - Lifetime
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08269087A (en) * | 1992-03-02 | 1996-10-15 | Fumio Yamauchi | New tetrapeptide and pentapeptide, their production and antihypertensive containing the same as active ingredient |
| US6232438B1 (en) | 1996-01-11 | 2001-05-15 | Nong Shim Co., Ltd. | Angiotensin converting enzyme inhibitors |
| WO2002055546A1 (en) * | 2001-01-16 | 2002-07-18 | Ajinomoto Co.,Inc. | Angiotensin converting enzyme inhibitors |
| EP1352911A4 (en) * | 2001-01-16 | 2007-11-28 | Ajinomoto Kk | Angiotensin converting enzyme inhibitors |
| WO2004011016A1 (en) * | 2002-07-30 | 2004-02-05 | Tropicana Products, Inc. | Cardiovascular health enhancement with soy fortified orange juice compositions |
| JP2007326790A (en) * | 2006-06-06 | 2007-12-20 | Aomori Prefecture | Method for producing acetic acid-enzyme treated product of apios americana, peptide derived from apios americana and method for producing the same |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0725793B2 (en) | 1995-03-22 |
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