JPH04154798A - Physiologically active peptide and its salt - Google Patents

Abstract

NEW MATERIAL:A peptide (salt) expressed by the formula (X is H or peptide (salt) expressed by the formula, H-Val-Leu-Pro-Ile-Pro-Gln-Gln). USE:A hypotensive agent having an inhibitory activity against angiotensin converting enzyme and a glucagon enhancing activity. PREPARATION:For example, a thermolysin is added to an aqueous solution of human milk beta-casein to dissolve in the solution and pH of the solution is controlled to 7.0 by NaOH. The solution is retained at 37 deg.C for 3hr, treated at 100 deg.C for 10min to inactivate the enzyme and the supernatant is collected by centrifugal separation. The supernatant is subjected to high-performance liquid chromatography and eluted by 0-50% acetonitrile concentration gradient to collect 25% acetonitrile elution fraction, and then the fraction is purified by repeating the fractionation under the same conditions to provide the peptide having the formula.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to novel peptides and salts thereof.

The peptide of the present invention has angiotensin converting enzyme inhibitory activity and glucagon enhancing activity, and is useful as a medicine such as a hypotensive agent.

[Conventional technology]

It is known that proteins contained in foods have not only nutritional effects but also various physiological activities.

For example, it is reported in Japanese Patent Publication No. 60-23085 and Japanese Patent Publication No. 60-2 that a peptide having a blood pressure lowering effect can be obtained by enzymatically decomposing casein, which is a milk protein.
It is disclosed in Japanese Patent Publication No. 3086, Japanese Patent Publication No. 60-23087, etc.

It has recently been confirmed that peptides obtained by enzymatically decomposing various proteins have various physiological activities, and the amino acid sequences of food-derived proteins are presumed to have potential biological functions.

The present inventors have discovered a novel peptide having a hitherto unknown sequence from the enzymatically decomposed product of human milk β-casein, and have completed the present invention.

[Problems solved by the invention]

An object of the present invention is to provide a novel peptide present in an enzymatic hydrolyzate of human milk casein, which has an angiotensin converting enzyme inhibitory activity and a glucagon activity enhancing effect.

[Means to solve the problem]

The peptide of the present invention is present in the enzymatic hydrolyzate of human milk β-casein and has a structure represented by the following formula (I).

X-Val-Val-Pro-Tyr-Pro
-G l n-Arg-01 ((I) (However, X is H or tl-Va 1-Leu-Pro-14!
This is a peptide represented by e-Pro-G 1 n-G l n.

The amino acid sequence of human milk β-casein is already known;
Among them are Va j! -Leu-Pro-Ile-
Pro-G ln-G1 n-Va l -Leu
The peptide sequence -Pro-Tyr-Pro-Vai-Arg was reported [the underlined amino acids are different from the substance of the present invention. R, Greenberg.

M.L., Groves, H.J., Dower, J.
Bkol, Chew, nL5132 (I984
)) The present inventors obtained a peptide by subjecting human milk β-casein to enzymatic degradation with trypsin or thermolysin, and determined the primary structure of this peptide. It was discovered for the first time that this peptide does not match the entire amino acid sequence and is a novel peptide.

Among the above peptides (I), H-Va ji! -Va 1-Pro-Tyr-Pr
o-G 1 n-Arg-OH (formerly obtained by decomposing β-casein with thermolysin, H-Va l -Leu-Pro-I 1 e-Pro
-G l n-G l n-Va Q -Va 12-
) Pro-Tyr-Pro-G l n-Arg
-Off (m) is obtained by decomposing human milk β-casein with trypsin. Furthermore, these peptides can also be prepared using commonly known chemical synthesis methods for peptides.

When human milk β-casein is prepared by enzymatic hydrolysis, it can be obtained by the following method.

Human milk whole casein was processed using 5ephadex G-150 under the conditions of Nagahama et al. [Journal of Dairy Science (
J, Dairy 5ci) Magazine 53136-145.19
70), enzymatically decompose with thermolysin or trypsin, inactivate the enzyme by heating, and then centrifuge to obtain a supernatant. This supernatant was mixed with octadecylsilyl (0
05) Column (Cosmosil 5C+s+ 250
Reversed phase high performance liquid chromatography (l
Purify by (PLC). The target peptide is 0
．． Upon development with a linear gradient of acetonitrile containing 1% trifluoroacetic acid, peptide (n)
Eluted with acetonitrile concentration, peptide (m) was 3
Elute at 6% acetonitrile concentration. Both fractions are rechromatographed under the same conditions to obtain purified peptide.

When it is prepared by chemical synthesis, it can be synthesized by the following method.

The SAM2 peptide synthesizer manufactured by BioSearch was used as a synthesis apparatus, and the desorption of the peptide from the resin as a carrier and the removal of the protecting group were carried out in anhydrous hydrogen fluoride containing 10% ansole at a temperature of 0°C. After distilling off the hydrogen fluoride, which is done by stirring for 1 hour, the resin is washed with ether and the peptides are extracted with 30% acetic acid. The peptides obtained by the extraction are subjected to gel filtration using a Biogel P-2 column (2.6 x 80 cm) equilibrated with 30% acetic acid, and then purified by reversed-phase high performance liquid chromatography using octadecylsilyl (2 x 25 Cl). The peptide of interest was developed with the formula (If
) is eluted, and at a concentration of 36% acetonitrile, the peptide of formula (I[I) is eluted from the column. Both peptides can thereby be obtained.

The peptide thus obtained can be identified by sequence analysis using an amino acid sequencer and amino acid composition.

Furthermore, the peptide of the present invention forms salts with acetic acid, hydrochloric acid, etc. The present invention also includes these salts.

All of the peptides of the present invention have angiotensin converting enzyme inhibitory activity and glucagon enhancing activity. These activities are confirmed as follows.

■ Angiotensin converting enzyme inhibitory effect Using angiotensin converting enzyme extracted from rabbit lung acetone powder, the method of Cushman and Cheurlg (Bioc
hem, Pharol, 20.1637-164
8 (I971)).

IC of peptide (II) of the present invention. is 20 μH, ICS of peptide (DI). Both peptides exhibited strong inhibitory activity against angiotensin convertase.

■ Enhancement of glucagon activity The protein catabolism of glucagon is measured using the amount of urea synthesis in primary cultured hepatocytes as an indicator.

2x105 cells/well in a 24-well dish
After culturing for 24 hours in the presence of 10% serum, culture is continued for an additional 24 hours in a serum-free medium. After that, ^
Two pre-incubations were carried out for 30 minutes in rg freeMIEM (HachiFM), and then the samples were added to the prescribed concentrations in HachiF.
Culture with M or APM+glucagon (I0-''M) for 6 hours. Take 200 μl of the cultured medium and quantify the amount of urea in the medium by the diacetyl monooxime method.

In the case of glucagon alone, the amount of urea synthesized was 8 μg/−. 23gg/d with addition of peptide (II)
, the addition of peptide (I[I) showed a value of 18 gg/lri, both of which enhance glucagon catabolism.

EXAMPLES Below, examples will be shown and the present invention will be further explained in detail.

Example 1 (Val - Va 1 from human milk β-casein
-Preparation of -Pro-Tyr-Pro-G ln-^rg) Thermolysin was added to an aqueous solution of human milk β-casein dissolved at a concentration of 10 μg/Id to a concentration of 100 μgod, dissolved, and the pH was adjusted to 7.0 μg with NaOH. Adjusted to 0.

The aqueous solution was kept at 37°C for 3 hours and then heated to 100°C.
The enzyme was inactivated by holding at C for 10 minutes. Thereafter, a supernatant was obtained by centrifugation, and this supernatant was subjected to IIPLc. HPLC uses Cos+*osil 5C, column (
250x20m, manufactured by Nacalai Tesque), 0 to 5
10 ae under linear gradient conditions of 1%/min with 0% acetonitrile gradient (containing 0.1% trifluoroacetic acid)
It was melted at a flow rate of /min. The peptide of interest was eluted in both fractions eluted with 25% acetonitrile, shown as peak A in FIG. This fraction was sieved and then eluted again under the same concentration gradient conditions and at the same flow rate.

When the obtained peptide was analyzed with a peptide sequencer, Va 1-Va j2-Pro-Tyr-Pr
o-G j! It had the sequence n-^rg. Furthermore, this peptide was subjected to silica gel thin layer chromatography. As a developing solvent, n-butanol:acetic acid:pyridine:water=
When expanded using 15=3:10:12, the corrected value was 0.41.

Example 2 (Val-Leu- from human milk β-casein)
Pro-T le-Pro-G l n-G l n
-Va 1-Va 1-Pro-Tyr-Pro-G
Preparation of l n-^rg) 100μs/m! Trypsin was added to dissolve the mixture, and the pH was adjusted to 7.0 with NaOH. The following operations were performed in the same manner as in Example 1.

The target peptide is 36, shown as peak B in Figure 2.
% acetonitrile. Both fractions were rechromatographed in the same manner as in Example 1 to obtain a purified peptide. This peptide is transferred to a protein sequencer.
According to the analysis, Va 1. -Leu-Pro-I
le-Pro-G j! n-G 1 n-Val
-Va 1-Pro-Tyr-Pro-G j! n-
It was confirmed that it has the sequence ＾rg. Furthermore, this peptide was subjected to silica gel thin layer fluorography. n-butanol:acetic acid:pyridine:water=ts as developing solvent
: When developed using 3:10:12, the Rf value showed 0.41.

Example 3 (Va 1-Va 1-Pro by chemical synthesis
-Tyr-Pro-G j! n-^rg and Val
-Leu-Pro4ffie-Pro-Gj! n-G
l n-Va l -Va 1-Pro-Tyr-Pr
Preparation of o-G 12 n-^rg) Synthesis was performed using a SAM2 peptide synthesizer (manufactured by Biosearch) according to the protocol of the same device.

That is, 0.3+g mol of t-Boc-＾rg per 1g
3 g of acyloxymethyl resin bound with (Tos) was set in the reaction vessel of the peptide synthesizer, and 45% (v/
v)) The t-Boc group was removed by contacting for 20 minutes with a deblocking solution containing lifluoroacetic acid, 2.5% (v/v) anisole, and 52.5% (v/v) dichloromethane. After washing with dichloromethane, the resin was neutralized with dichloromethane containing 10% (V/V) diisopropylethylamine and washed with dichloroquine. Then 6III
lol's t-Boc-Pr1 and 6m++wo 1.
of diisopropylcarbodiimide (6.7 times the theoretical equivalent of each) in a dichloromethane/dimethylformamide mixture of 30 af for a period of time at room temperature. After sequentially washing with dimethylformamide and dichloromethane, deblocking was performed in the same manner as above, and the same procedure was followed↓from the C-terminal side, t-Boc-G Q n, t-Bo
c-Pro, t-Boc-Ty (Cffiz-Bzl
), t-Boc-Pro+ t-Boc-Vaf+
t-Boc-Vaj! are sequentially combined, t-Boc
-Va l -Va 1-Pro-Try (c 1
t-Bz 12 )-Pro-G j! n-Arg(
Tos) resin was obtained (Resin 1).

173 of this resin was fractionated, and after deblocking in the same manner as above, 2+u+ol of t-Boc-G 1
n+t-Boc-Gj! n+ t-Boc-Pro+
t-Boc4ffe, t-Boc-Pr.

t-Boc-Leu+t-Boc-Va 12 are sequentially combined to form t-B.

Vaj! -J, eu-Pro-IleJro-GI! ,
n-Gin-Val, -Vai! Pro-Tyr (C
j! , Bzl)-Pro-Gfn-Arg(Tos)-
Tree decomposition (resin ■) was obtained.

3 After reacting Resin I and ■ in anhydrous hydrogen fluoride containing 10% anisole at 0°C for 1 hour,
Hydrogen was removed by distillation and washing with ether was performed.

2 lO% from the resulting mixture of peptide and resin
The peptides were extracted with acetic acid and freeze-dried to obtain approximately 50 h+g of crude peptides 1 and 2, respectively.

Dissolve crude peptide 1 in 0.1% trifluoroacetic acid 1
After that, octadecylsilane (005) column (Co
High performance liquid chromatograph (M2
O3 type, manufactured by Nippon Waters Co., Ltd.), a linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid (θ ~ 30%/30 minutes), 10d! It unfolded in 7 minutes. Target peptide 1 has acetonitrile concentration of approximately 25%
The substance obtained in this way, which was eluted at -SheaI! , -0-Pro-Tyr-Pro-
The fact that it was G 12 n-Arg was also confirmed by amino acid analysis and protein sequencer (Applied Biosystems 477^).

Moreover, when crude peptide 2 was eluted using a linear concentration gradient of acetonitrile (0 to 40% over 740 minutes) in the same manner as above, it was eluted at an acetonitrile concentration of about 36%. The peptide thus obtained was Val-Leu-Pro.
-14! e-Pro-G iL n-G l n-V
a l -Va l -Pro-Tyr-Pro-G
1 n-Arg was confirmed by amino acid analysis and protein sequencer.

〔Effect of the invention〕

By carrying out the present invention, novel peptides having various physiological activities such as angiotensin converting enzyme inhibiting action and glucagon enhancing action can be provided. This novel peptide is useful as a medicine such as an antihypertensive agent.

[Brief explanation of the drawing]

Figure 1 shows human milk β that has been enzymatically degraded with thermolysin.
HP of casein with Cosmosil 5CH column
The target peptide c formula (■) is contained in the A portion, which shows the LC pattern. Figure 2 shows HP analysis of human milk β-casein on a Cosmosil 5C+s column after enzymatic digestion with trypsin.
The target peptide (2) is contained in peak B shown in the LC pattern.

Claims (1)

[Claims] A peptide represented by the following general formula (I) or a salt thereof X-Val-Val-Pro-Tyr-Pro-Gln
-Arg-OH (I) (where X is H or H
-Val-Leu-Pro-Ile-Pro-Gln-
peptide expressed by Gln)