JPH0499798A - New peptide and its salt - Google Patents

Abstract

NEW MATERIAL:The peptide of formula I (X is Leu or Phe; Y is Ser or Ala; Z is Val or Leu) or its salt. EXAMPLE:The peptide of formula II. USE:Useful as a hypotensor, etc., having ileum constriction action, canine mesenteric artery relaxing action, angiotensinase inhibiting action, etc. PREPARATION:The objective peptide can be produced e.g. by decomposing serum albumin with trypsin, deactivating the enzyme, centrifuging the product, subjecting the obtained supernatant to reversed-phase high-performance liquid chromatography (HPLC) with octadecylsilyl column, eluting with acetonitrile containing 0.1% trifluoroacetic acid under a linearly varying concentration gradient and finally purifying the obtained active fraction by HPLC treatment using a phenyl column and a cyanopropyl column.

Description

[Detailed Description of the Invention] The present invention relieves the guinea pig ileal constriction effect and canine mesenteric artery prostaglandin F2α-induced constriction, and furthermore inhibits angiotensin converting enzyme activity and lowers blood pressure. It relates to a novel peptide having the following properties. The peptide of the present invention is useful as a medicine such as a blood pressure lowering agent.

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

For example, by subjecting casein, which is a milk protein, to enzymatic decomposition, a peptide having a blood pressure lowering effect can be obtained, as disclosed in Japanese Patent Publication No. 60-23085 and Japanese Patent Publication No. 60-23.
It is disclosed in Japanese Patent Publication No. 086, Japanese Patent Publication No. 60-23087, etc.

It has recently been confirmed that peptides obtained by enzymatically decomposing various proteins have various physiological activities1, and it is assumed that the amino acid sequences of food-derived proteins have potential biological functions. .

In the process of researching peptides derived from bovine serum albumin, the present inventors focused on the amino acid sequence of serum albumin, discovered a new peptide with physiological activity from this amino acid sequence, and completed the present invention. Ta.

The present invention is a novel peptide derived from the amino acid sequence of serum albumin, which releases ileal contractility and canine mesenteric artery prostaglandin F2α-induced contraction, and has angiotensin converting enzyme inhibitory activity and blood pressure. Our goal is to provide substances that have physiological activities such as depressing effects.

The novel peptide of the present invention exists in the primary structure of bovine serum albumin and corresponds to residues 208 to 216, and also exists in the primary structure of bovine serum albumin and corresponds to residues 210 to 219 of human serum albumin. It also corresponds to residues 208 to 216 of serum albumin.The peptide derived from bovine serum albumin is Aia-
Leu-Lys-A 1 a-Trp-3er-sial
-AlaArg, and the human serum-derived peptide is Aj2aPhe-Lys-A I! , a-Trp-8i! , a-Val-Ala-Arg, the peptide derived from fi is A11a-Phe-Lys-^4
2 a-Trp-5erLeu-^ffia-Arg.

In order to obtain the peptide of the present invention, it is possible to obtain it by chemical synthesis, and it is also possible to obtain a purified substance by subjecting serum albumin to enzymatic decomposition and further performing high performance liquid chromatography treatment. When performing 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 performed at a temperature of 0°C in anhydrous hydrogen fluoride containing 10% anisole. This is done by stirring for 1 hour under these conditions.

After distilling off the hydrogen fluoride, the resin is washed with ether and the peptides are extracted with 309A acetic acid.

The peptides obtained by extraction were subjected to gel filtration using a Biogel P-2 column (2,6 x 80 cm) equilibrated with 30% acetic acid, and then subjected to octadecylsilyl (ODS).
Column (Cosmosil 5C+'s, 2X25cm
) and purified by reversed-phase high-performance liquid chromatography. The target peptide was eluted from the column at a concentration of 30% acetonitrile during development using a linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid to obtain the target peptide.

Furthermore, when serum albumin is prepared by enzymatic hydrolysis, it can be obtained by the following method.

After decomposing serum albumin by trypsin and inactivating the enzyme by heating, centrifugation is performed to obtain a supernatant. This supernatant is subjected to IPLc treatment (under the same conditions as the column described above).

The desired fraction is eluted at a concentration of 30% acetonitrile.

Furthermore, this fraction was subjected to HPLC treatment using a PhenyJ column and a cyanopropyl column to obtain the target peptide.

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, and the like. The present invention also includes these salts.

The novel peptide thus obtained has a guinea pig ileal contraction test, a canine mesenteric artery relaxing effect, and a hypotensive rat blood pressure lowering effect. These physiological activities are confirmed as follows.

■ Ileal contraction effect Ileal muscle sections taken from guinea pigs weighing 300 to 500 g were processed according to the method written by Takagi et al. (edited by Takagi et al., Pharmacology Experiments, pp. 94-99, 1972, Nanzando), and used for measurement. provided. One end of the specimen is connected to an isobencial transducer via a thread, and the other end is
This peptide was fixed in the Magnus tube of the patient, and the tension of muscle contraction was electrically recorded.
. It was a value.

■ Canine mesenteric artery relaxation effect A spiral section of canine mesenteric artery was connected to an isobencial transducer in a Magnus tube in the same manner as in the guinea pig ileal contraction test. 0.5μH prostaglandin F2
This peptide showed a relaxing effect on the mesenteric artery constricted by α. EDs at that time. The value was on the order of 1.1μ.

■ Angiotensin converting enzyme inhibitory effect Using angiotensin converting enzyme extracted from rabbit lung acetone powder, the method of Cushman and Cheung [Bioch
em, Pharm, 20.1637-1648
(1971)]. I of this peptide
Cs. The value was 3.8 μ-.

EXAMPLES The present invention will be further explained in detail by way of examples below.

Example 1 (A j1! a-Le from bovine serum albumin
u-LysA I1a-Trp-3er-Va 1. −
81 Preparation of a-Arg) Trypsin was added to an aqueous solution containing live serum albumin at a concentration of 10 mg/ail to give a concentration of 200 mg/1 ail, dissolved, and purified with NaOH.
H was adjusted to 7.8.

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 HPLC.

For HPLC, Cosmosil 5C+s column (25
0x20mm.

Using a 0-60% acetonitrile concentration gradient (containing 0.1% trifluoroacetic acid),
Elution was carried out at a flow rate of 10 m/min under linear concentration gradient conditions of 10 m/min. The peptide of interest was eluted in both regions indicated by the opening in FIG. Collect these two parts, then Cosmosi
H equipped with l 5Ph (150 x 4.6 W column)
It was subjected to PLC and eluted under the same concentration gradient conditions at a flow rate of 1IR1/min.

The target peptide was eluted at the position indicated by O in FIG. This fraction was collected and further coated with Cosmosil 5CN.
Subjected to HPLC using a column (250 x 4.6-m),
It was eluted under the same conditions as above. The peptide of the present invention was eluted at the position indicated by ↓ in FIG. When the obtained peptide was analyzed using a peptide sequencer, Aj2aLeu
-Lys-A j2 a-Trp-5er-Va l
-A 1 a-Arg. In addition, when the amino acid composition ratio of the peptide was analyzed using an amino acid analyzer, it was found that A1a:Arg:Leu:Lys:Ser
: Trp : Va 1 is 3:1:1:1:1:1:
One by one was detected, which matched the theoretical value. Furthermore, this peptide was subjected to silica gel thin layer chromatography. As a developing solvent, n-butanol:acetic acid:pyridine:water-15:3:
When I used 10:12 and expanded it, the Rf value was 0.
．． It showed 33.

Both fractions were collected and further subjected to 1 (PLC) using a Cosmosil SCN column (250×4.6n+n+) and eluted under the same conditions as above. The peptide of the present invention was placed in a container at the position indicated by ↓ in FIG.

Using a male spontaneously hypertensive rat with a blood pressure below the knee of 400 g, a cannula was inserted into the carotid artery under urethane anesthesia, and this cannula was connected to a pressure transducer for recording on a polygraph.

The antihypertensive agent of the present invention prepared in Example 1 was administered through the femoral vein, and the decrease in blood pressure was observed. It was observed that the blood pressure of 160 mm) Ig decreased to 130 mn+Hg upon administration of 5111 g/kg.

Example 2 (A 1 a-Leu-Lys-A by chemical synthesis
j2 aTrp-5er-Va f -A I! ,a-
Preparation of Arg) SAM2 peptide synthesizer (Bios
(manufactured by EARCH Inc.) according to the protocol of the same device.

That is, 0.3 mmof2 t-Boc-Arg (
2 g of acyloxymethyl resin bound with Tos) was set in the reaction vessel of a peptide synthesizer, and the reaction volume was 45% (ν/v).
Trifluoroacetic acid, 2.5% (v/v) anisole, 5
The L-Boc group was removed by contacting with a debrotter solution containing 2.5% (v/v) dichloromethane for 20 minutes. After washing with dichloromethane, the resin was neutralized with dichloromethane containing 10% (V/V) diisopropylethylamine.
, washed with dichloromethane. Then 4.0 mm
o 1(7) tBoc-A j2 a and 4.0m
20IR1 dichloromethane containing mof diisopropylcarbodiimide (6.7 times the theoretical equivalent of each);
The reaction was carried out in a dimethylformamide mixture for 2 hours at room temperature. After sequentially washing with dimethylformamide and dichloromethane, unreacted α-amino groups were acetylated in dimethylformamide containing 0.4M N-acetylimidazole. After washing this resin with dichloromethane, deblocking was performed in the same manner as above, and t-Boc-Va R, t-Bo
cSer(Bz1), t-Boc-Trp, t
-Boc-A jl! a, L-Boc-Lys(C
f-Z)+t-Boc-Leu, t-Boc-Aff
ia are sequentially combined, t-Boc-81. a-Leu
-Lys(CR-Z) 8Ra-Trp-5er(B
z 1 )-Va 12-A 1 a-Arg(Tos
)-resin was obtained.

This resin was reacted in anhydrous hydrogen fluoride containing 10% anisole at 0° C. for 1 hour, and then the hydrogen fluoride was distilled off and washed with ether. The peptide was extracted from the resulting mixture of peptide and resin with 10% acetic acid and freeze-dried to obtain about 200 μg of crude peptide.

After dissolving the crude peptide in 0.1% trifluoroacetic acid,
Octadecylsilane (ODS) column (Cosmosi
A linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid (from 0 to
50% 150 minutes, 1 Oai! /min. The target peptide was eluted at an acetonitrile concentration of about 30%. The material power obtained in this way 'A l a-L
eu-LysA j2 a-Trp-3er-Va 1
-Eight! The fact that it is a-^rg was determined by amino acid analysis and protein sequencer (Applied Biosystems 47).
7A) was confirmed.

By carrying out the present invention, a novel peptide has been created which has various physiological activities such as ileal contraction, canine mesenteric artery prostaglandin F2α-induced constrictive action, angiotensin converting enzyme inhibitory action, and blood pressure lowering action. can be provided. This novel peptide is useful as a medicine such as an antihypertensive agent.

[Brief explanation of drawings]

Figure 1 shows the Cosm of bovine serum albumin after enzymatic degradation.
HPLC pattern with osfl 5C,6 column is shown. The internal portion contains the desired peptide. Figure 2 shows the 1 (PLC pattern) fractionated using a Cosmosil 5Ph column. The target substance is eluted in the second part. Figure 3 shows the HPLC pattern fractionated using a Cosmosil 5CN column. The target substance is eluted in the second part. It has been eluted.

Claims (1)

[Claims] A peptide represented by the following formula or a salt thereof H-Ala-X-Lys-Ala-Trp-Y-Z-A
la-ArgOH (However, X represents Leu or Phe, Y represents Ser or Ala, and Z represents either Val or Leu.)