JPH051097A - Angiotensin i converting enzyme-inhibitory pentapeptide and its production - Google Patents

Abstract

PURPOSE:To provide an angiotensin I converting enzyme-inhibitory peptide available at a low cost with hardly any side effects and a suitable method for producing the peptide. CONSTITUTION:The tripeptide is an angiotensin I converting enzyme-inhibitory peptide, having a structure of Ile-Arg-Pro-Val-Gin and produced by separating and purifying the peptide from an extract solution obtained from viscera and muscles of fishes and shellfishes or a chemical synthetic method by using Fmoc- isoleucine, Fmoc-arginine, Fmoc-proline, Fmoc-valine and Fmoc-glutamine. Since the Ile-Arg-Pro-Val-Gin has angiotensin I converting enzyme 7-inhibitory ability, the blood pressure is reduced.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an angiotensin I converting enzyme inhibitory peptide and a method for producing the same. More specifically, the present invention relates to an angiotensin I-converting enzyme inhibitory peptide which is an enzyme that converts angiotensin II from angiotensin I, which works to increase blood pressure, and a method for producing the same.

[0002]

2. Description of the Related Art Hypertension is a condition in which the maximum blood pressure is 160 mmHg or higher, the minimum blood pressure is 95 mmHg or higher, or both. It is said that the number of patients in Japan is about 20 million, which is a disease with a high morbidity.

It is known that hypertension causes various complications over a wide range of organs such as cerebral hemorrhage, cerebral infarction, subarachnoid hemorrhage, angina, myocardial infarction, renal sclerosis, renal failure and retinal vein occlusion. Therefore, effective therapeutic agents are desired.

As one of the mechanisms for controlling blood pressure in the living body, there are the renin-angiotensin system which is a pressor system and the kallikrein-quinine system which is a hypotensive system. In the renin-angiotensin system, the enzyme renin is produced in renal glomerular cells (JG cells), and acts on angiotensinogen, which is a renin substrate in blood vessels, to produce angiotensin I. The enzyme that converts angiotensin I into angiotensin II is an angiotensin I converting enzyme, and the resulting angiotensin II acts on arterioles to cause contraction.

[0005] Angiotensin II also acts on the adrenal cortex, promotes synthesis and secretion of aldosterone, promotes reabsorption of sodium in the kidney, and maintains the body fluid volume. Angiotensin II thus raises blood pressure.

On the other hand, in the kallikrein-quinine system, kallikrein, which is a proteolytic enzyme, acts on kininogen, which is a substrate, to generate quinine. Kininase dilates blood vessels and lowers blood pressure, but kininase II
Undergo decomposition by. Kininase II is known to be the same substance as angiotensin I converting enzyme.

From the above, treatment of hypertension by inhibiting angiotensin I converting enzyme is considered, and to date, captopril, enalapril, alacepril,
Delapril and others are being developed. Peptides derived from casein, zein, gelatin, fish meat, etc. are also known to have an inhibitory effect on angiotensin I converting enzyme (JP-A-59-44324 and JP-A-64-324).
5497, JP-A 1-313498).

However, although captopril has a strong antihypertensive effect, it is expensive because it is produced by a synthetic method, and it is difficult to obtain it easily. Moreover, inadequate dosage results in renal dysfunction and hypotension. Further, it is said that the SH group existing in the molecule causes rash and taste abnormality. Peptides derived from casein, zein, fish meat, etc. are derived from natural products, which generate a lot of unused substances in the manufacturing process, which not only enormously costs to process them, but also causes environmental pollution due to disposal. I have a concern.

[0009]

The present invention provides an angiotensin I-converting enzyme inhibitor that is inexpensively available, has few side effects, and does not produce a new unused product during production, and a suitable method for producing the same. The purpose is to provide.

[0010]

Means for Solving the Problems The present inventors have completed the present invention as a result of various studies for solving the above problems. That is, the present invention provides Ile-Arg-Pro-Va derived from seafood viscera that is a by-product of seafood processing.
It is an angiotensin I converting enzyme inhibitory peptide having a structure of 1-Gln.

The present invention also provides the above-mentioned angiotensin I.
The converting enzyme inhibitory peptide is extracted from the viscera and muscles of seafood, or further, Fmoc-isoleucine and Fmoc
-A method of producing by solid phase synthesis using arginine, Fmoc-proline, Fmoc-valine, Fmoc-glutamine. The present peptide can be obtained by extracting water from the internal organs and muscles of seafood with water, heating, and then separating the extracted peptide-containing substance using various chromatographic techniques.

The peptide of the present invention was obtained by adsorbing the peptide in a water extract onto a packing material for reverse phase chromatography C ₁₈ (a silica carrier having an octadecyl group bonded), washing with water and eluting with a 15% acetonitrile solution. Minute cation exchanger CM
(Carboxymethyl group-bound carrier) was adsorbed, and the angiotensin I-converting enzyme inhibitory fraction among the fractions obtained by the stepwise elution method was further separated by reverse phase HPLC,
The active fraction is further obtained by separation by gel filtration HPLC.

As the fish species usable in the present invention, any fish species such as bonito, tuna, horse mackerel, mackerel, sardine, saury, yellowtail, thailand, flounder, flatfish, perch, salmon, herring, squid, octopus and the like can be used. As the internal organs, any of stomach, pyloric appendix, intestine, liver, testis, ovary, heart and the like may be used. Also, seafood muscles can be used.

Further, the present peptide includes Fmoc-isoleucine, Fmoc-arginine, Fmoc-proline and Fmoc-proline.
It can also be produced by a chemical synthesis method such as a solid-phase synthesis method using moc-valine and Fmoc-glutamine.

[0015]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 106.3 g of skipjack intestine was added with a 4-fold amount of water, and a homogenizer (Nissei AM-1 type) was used to cool the mixture under ice cooling.
Homogenized for 5 minutes at 000 rpm.

After autoclaving at 120 ° C. for 5 minutes, centrifugation was performed at 40,000 G for 40 minutes at 4 ° C., and the supernatant was concentrated, and the C ₁₈ packing material (Waters Bulk C ₁₈
(55-105 μm) 100 g was adsorbed on a simple column. After washing this column with 1435 ml of distilled water, the peptide was eluted with a 15% acetonitrile solution. By this operation, 4.2 g of peptide fraction was obtained.

The peptide fraction of the present invention is an accelerator CM (Water) equilibrated with a 10 mM phosphate buffer (pH 6.0).
s company) was added to a simple column packed with 75 g, and 750 ml of 10 mM phosphate buffer (pH 6.0), (pH 7.
0) 750 ml, (pH 8.0) 750 ml, (pH 9.
0) Elution with 750 ml. Of these, Angiotensin I
In order to further purify the pH 8.0 elution fraction having a strong ability to inhibit the converting enzyme, separation was performed by reverse phase HPLC. This HPLC
The conditions are shown in the following HPLC condition 1.

HPLC condition 1 Column RP-18 (e) 1 × 25 cm UV detection at Merck, UV 210 nm Flow rate 4 ml / min Eluent A: 0.05% TFA B: 0.05% TFA 50% acetonitrile A 100% to 800 min Later, a linear gradient such that A50% and B50%

Under the above condition 1, a peptide having an angiotensin I converting enzyme inhibitory ability has a retention time of 13
It was eluted at 3 minutes. The peptide was further purified by HPLC for further purification. The conditions at this time are shown in the following HPLC condition 2.

HPLC condition 2 column RP-18 (e) 4 mm × 25 cm UV detection at Merck, UV 210 nm flow rate 1.0 ml / min eluent A: 0.05% TFA B: 0.05% TFA 25% acetonitrile A 100% After 800 minutes, a linear gradient such that A50% and B50% are obtained.

Under the above condition 2, the retention time 26
Strong angiotensin I-converting enzyme inhibitory activity was observed at 2 minutes (peak 1 in the chart shown in FIG. 1). In order to further purify this peak, separation was performed under the following HPLC condition 3.

HPLC condition 3 column Asahi Pack GS-320 7.5 mm × 5
0 cm Asahi Kasei Co., Ltd. Detection UV 210 nm Flow rate 1.0 ml / min Eluent 50 mM ammonium acetate solution

Under this condition, the peptide having an angiotensin I converting enzyme inhibitory ability has a retention time of 16.
It was eluted at 6 minutes (Peak 2 in Chart 2 shown in FIG. 2). This peak was further subjected to HPLC under HPLC condition 3, and a single peak was obtained at a retention time of 16.6 minutes. Therefore, this peak was subjected to Edman decomposition reaction using a gas phase protein sequencer (Model-470A) manufactured by Applied Biosystem and online to identify the PTH amino acid obtained in each cycle. As a result, SEQ ID NO: 1 (sequence: Ile-Ar
It was found to have the structure of g-Pro-Val-Gln).

The angiotensin I-converting enzyme inhibition is measured by the method of Kashman et al. [Biochemical Pharmacology 20: 1637-1648 (1971)].
The method of Maruyama et al. [Agricultural Biological Chemistry Vol. 46, No. 5, 1393-13]
94 (1982)].

That is, a test tube containing 30 μl of the peptide aqueous solution of the present invention, L-hypril histidyl leucine (manufactured by Sigma) as an enzyme substrate, and NaCl containing pH 8.3.
250 ml of borate buffer (1) was added and preincubated at 37 ° C. for 10 minutes. Then, 100 μl of angiotensin I converting enzyme-containing solution was added to start the enzymatic reaction. At this time, the concentration of borate buffer is 0.1M, L
-Hypryl histidyl leucine concentration is 5 mM, NaCl
The enzyme activity is 300 mM, and the enzyme activity without inhibition is 8 mU. After reacting with shaking at 37 ° C. and pH 8.3 for 30 minutes, 250 ml of 1N HCl was added,
The reaction was stopped. Ethyl acetate (1.5 ml) was added, and the mixture was shaken for 15 seconds to extract hippuric acid generated by the enzymatic reaction.
Centrifuge at 0 rpm for 10 minutes and 1.0 ml of ethyl acetate layer
Was collected in a test tube. Ethyl acetate was heated in a hot dry bath at 120 ° C. for 30 minutes to completely remove it, and then left at room temperature for 5 minutes. Then, 1.0 ml of H ₂ O was added, and the amount of hippuric acid produced was determined by measuring the absorbance at 228 nm. The liquid containing angiotensin I converting enzyme used for the enzyme reaction is rabbit acetone powder (manufactured by Sigma).
1 g of 0.1 M borate buffer (pH 8.3) 10 ml
Dissolve in water and stir well, then 40 at 40,000G at 4 ℃.
It was prepared by diluting the supernatant after centrifugation for 0.1 minutes with 0.1 M borate buffer.

The inhibition rate was calculated using the following formula. A: Absorbance upon addition of distilled water (228 nm) B: Absorbance upon addition of inhibitor (228 nm) According to this method, the peptide inhibited the activity of angiotensin I converting enzyme by 47% per 1.0 μg of protein.

Example 2 Peptides of the present invention are Fmoc- (9-fluorenylmethyloxycarbonyl) amino acids (Fmoc-isoleucine, Fmoc-arginine, Fmoc-proline, F
It was synthesized by a solid-phase synthesis method using moc-valine, Fmoc-glutamine). This peptide is YMC-ODS-
R5 column (manufactured by YMC Co., Ltd.) with 0.1% T
Analysis is performed by a 25-minute linear gradient from an initial solution of an aqueous FA (trifluoroacetic acid) solution to a final solution of 70% acetonitrile solution of 0.1% TFA (flow rate 1.
As a result of 0 ml / min, detection UV 210 nm), a purity of 98% was shown. As a result of measuring the angiotensin I converting enzyme inhibitory ability using this substance, it was the same value as that extracted from the bonito intestine.

[0028]

INDUSTRIAL APPLICABILITY According to the present invention, an angiotensin I-converting enzyme inhibitory peptide having an action of suppressing an increase in blood pressure can be obtained from the internal organs of seafood that has been conventionally discarded.

[Sequence list]

SEQ ID NO: 1 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin of organism name: Katsuwonas pelami
s) Arrangement Ile Arg Pro Val Gln 1

[Brief description of drawings]

FIG. 1 shows a chart in Example 1 in which a fraction having a strong ability to inhibit angiotensin I converting enzyme was separated under HPLC condition 2.

FIG. 2 shows a chart in which peak 1 in FIG. 1 is further separated under HPLC condition 3.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location // C07K 99:00

Claims (3)

[Claims] 1. Ile-Arg-Pro-Val-Gl
An angiotensin I converting enzyme inhibitory peptide having a structure of n. However, Ile is isoleucine, Arg is arginine, Pro is proline, Val is valine, and Gln is a glutamine residue. 2. An angiotensin I conversion having a structure of Ile-Arg-Pro-Val-Gln, characterized in that the extract obtained by extracting from the internal organs and muscles of seafood with a solvent is separated and purified. A method for producing an enzyme-inhibiting peptide. 3. Fmoc-isoleucine, Fmoc-arginine, Fmoc-proline, Fmoc-valine, F
A method for producing an angiotensin I converting enzyme inhibitory peptide having a structure of Ile-Arg-Pro-Val-Gln, which comprises performing solid phase synthesis using moc-glutamine.