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

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

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antihypertensive agent containing a novel tripeptide as an active ingredient and a process for producing the novel tripeptide.

[0002]

2. Description of the Related Art Hypertension is broadly classified into etiologically clear cause of elevated blood pressure (symptomatic hypertension) and unknown (hypertensive hypertension). Although symptomatic hypertension can cure the hypertension by curing the causative disease, it is difficult to treat the cause directly with essential hypertension. Conventionally, the renin-angiotensin system (hereinafter,
Abbreviated as RA system. ) Is considered to be one of the important factors of essential hypertension.
By inhibiting the activity of angiotensin converting enzyme (hereinafter abbreviated as ACE) that plays a central role in the system, R. Attempts have been made to regulate the A system to regulate essential hypertension. Examples of such substances having ACE activity inhibition include L-proline derivatives [M. A. Ondetti, B.A. Rubin et
al; Science, 196, 441 (197).
7)] and compounds based thereon are known, and in the case of a substance derived from a natural product, a venom-derived bradykinin enhancer (C-terminal is Pro) [S. H. Ferria,
D. C. Bartelt et al; Biochem
istry, 9, 3583 (1970)], six peptides derived from collagenase digestion products of gelatin (Ala-Hyp at the C-terminus) [G. Oshima, H .; Shima
bukuro et al; Biochim. BioP
hs, Acta, 566, 128 (1979)], a peptide derived from a tryptic digest of bovine casein (C-terminal is
Gly-Lys) [S. Maruyama, H .; Suz
uki; Agric. Biol. Chem, 46, 13
93 (1982)] and the like are known. In the case of food, Suzuki et al. Have found inhibition of ACE activity in soybeans, teas, shellfish and fruits [Takeo Suzuki, Nobuko Ishikawa et al .; Agriculturalization, 5
7, 1143 (1983)]. However, all of these substances derived from natural products have only been confirmed to be effective by intravenous administration, and the pharmacological effect by oral administration is unknown, and although it has been a long time since the invention, it is still a drug. There is no report that development is progressing.

[0003]

An object of the present invention is to provide a novel tripeptide, a method for producing the same, and an antihypertensive agent containing the same.

[0004]

Means for Solving the Problems As a result of intensive studies for solving the above-mentioned problems, the present invention has revealed that the novel peptide of the present invention obtained from a decomposition solution of proteolytic enzymes of sardine muscle and soybean The inventors have found that it has a lowering action and have completed the present invention. That is, the present invention is 23 novel tripeptides having an L-amino acid sequence represented by:

(2) The product obtained by treating sardine muscle with a proteolytic enzyme is filtered, and the components of the filtered component that have passed through the semipermeable membrane are sequentially treated with a strongly acidic cation exchange resin,
It is characterized in that it is fractionated by gel filtration, ion-exchange gel filtration, reverse-phase high performance liquid chromatography, and fractions containing components having angiotensin converting enzyme inhibitory activity are obtained from the fractions obtained by each treatment. The new 2 above
A method for producing three types of tripeptides. (3) An antihypertensive agent containing the novel tripeptide as an active ingredient. 27 novel tripeptides having an L-amino acid sequence represented by:

(5) The product obtained by treating soybean with a proteolytic enzyme is filtered, and the components of the filtered component that have passed through the semipermeable membrane are sequentially filtered with a strong acid cation exchange resin, gel filtration, Ion-exchangeable gel filtration, fractionated by reverse-phase high performance liquid chromatography, from the fraction obtained for each treatment to obtain a fraction containing a component having angiotensin converting enzyme inhibitory activity, A method for producing 27 novel tripeptides. (6) The present invention relates to an antihypertensive agent containing the novel tripeptide as an active ingredient. Hereinafter, the present invention will be described in detail. The novel tripeptide of the present invention is (The above 23 kinds, each symbol in the formula of tripeptide represents each amino acid unit of the amino acid sequence in peptide chemistry.)

[0007] (The above 27 kinds, each symbol in the formula of tripeptide represents each amino acid unit of the amino acid sequence in peptide chemistry.)
It is a novel tripeptide having an L-form amino acid sequence represented by and has the property of white powder at room temperature.

Examples of the method for producing the novel tripeptide described above include a method for chemically synthesizing the tripeptide or a method for separating and purifying from the degradation solution of sardine muscle and soybean proteolytic enzymes. In the case of chemically synthesizing the novel tripeptide of the present invention, it can be carried out by an ordinary synthetic method such as a liquid phase method or a solid phase method, but preferably, it is a solid phase support of a polymer based on the solid phase method. It is preferable that the amino acids of L-form corresponding to the amino acid residues of the tripeptide are sequentially linked to the body from the C-terminal side (carboxyl-terminal side) by peptide bonds.
Then, the synthetic tripeptide thus obtained is
After cleaving from the polymeric solid-phase support with trifluoromethanesulfonic acid, hydrogen fluoride, etc., the protecting group of the amino acid side chain was removed, and high-performance liquid chromatography using a reversed-phase column (hereinafter, HPLC) It is abbreviated.) And the like.

The novel tripeptide of the present invention can be separated and purified from the degradation solution of proteolytic enzymes of sardine muscle and soybean. In that case, the 1991 Annual Meeting of the Japanese Society of Agricultural Chemistry (Kyoto), P183. 3AP1
According to the method of No. 3, it can be performed as follows, for example. The sardine muscle part and soybean containing the above novel tripeptide are taken out, and a suitable solvent (for example, water, Tris-HCl buffer,
After sufficient homogenization in a neutral buffer such as phosphate buffer), hydrolyze. Hydrolysis is performed according to a conventional method. For example, when hydrolyzing with a proteolytic enzyme such as pepsin, the sardine muscle homogenate and soybean homogenate are further hydrolyzed if necessary, then heated to the optimum temperature of the enzyme to adjust the pH to an optimum value, Add enzyme and incubate. Then, after neutralizing as necessary, the enzyme is deactivated to obtain a hydrolyzed solution. The hydrolyzate is filtered with a filter paper and Celite to remove insoluble components, and the resulting filtrate is filtered with a semipermeable membrane such as cellophane to a suitable solvent (for example, water, Tris-hydrochloric acid buffer). Neutral buffer such as liquid phosphate buffer)
The solution containing the components that have passed through the semipermeable membrane with the components in the filtrate after being sufficiently dialyzed in a strong acidic cation exchange resin (for example,
Dowex 50W made by Dow Chemical Co.)
A fraction containing a component having angiotensin converting enzyme (hereinafter abbreviated as ACE) inhibitory activity was obtained from the adsorbed and eluted fraction, and the obtained ACE inhibitory activity fraction was subjected to gel filtration (for example, Sephadex manufactured by Pharmacia). G-
25), and the obtained ACE inhibitory activity fraction was fractionated by cation exchange gel filtration (for example, SP-Sephadex C-25 manufactured by Pharmacia) to obtain the obtained ACE inhibitory activity fraction. The image is further HP
It can be performed by fractionation by LC (reverse phase high performance liquid chromatography).

As the fish muscles and legumes used in the method for producing the novel tripeptide of the present invention, any fish muscles and legumes may be used as long as the object of the present invention can be achieved, but preferably sardines and soybeans. It is better to use. The novel tripeptide of the present invention obtained as described above does not induce antibody production and does not cause anaphylactic shock even after repeated intravenous administration. Further, the novel tripeptide of the present invention has a sequence structure of only L-amino acids, and its molecular size shows that
After administration, it is promptly absorbed in the intestinal tract without being decomposed by proteases in the body, and exerts its hypotensive effect, so toxicity is extremely low and safety is extremely high (LD
₅₀ ) 5000 kg / kg; rat oral administration). The novel tripeptide according to the present invention is an injection, tablet, capsule, granule,
It can be adjusted to powder and the like. The method of administration generally includes injection into mammals having ACE (eg, humans, dogs, rats, etc.), or oral administration. The dose is, for example, 1 kg of animal body weight.
The amount of this tripeptide is 0.01 to 10 mg per unit. The frequency of administration is usually about 1 to 4 times a day, but can be appropriately adjusted depending on the administration route. The novel tripeptide according to the present invention has an excellent angiotensin converting enzyme inhibitory action, and exhibits a blood pressure lowering action and a bradykinin inactivation inhibiting action. Therefore, essential hypertension, renal hypertension, prophylactic and therapeutic agents for hypertension such as adrenal hypertension, useful as a diagnostic agent for these diseases and as a blood pressure lowering agent used in various pathological conditions, further organs for congestive heart failure. It has normalization of circulation and improvement of long-term prognosis (life extension effect), and is useful as a therapeutic agent for heart failure.

EXAMPLES Hereinafter, the present invention will be described in more detail by describing production examples and test examples as examples.

Production Example 1 [Production of Novel Tripeptide from Sardine Muscle] 1 L of deionized water was added to 500 g of sardine muscle and homogenized, and then the pH was adjusted to 2.0 with 1N hydrochloric acid, and pepsin (manufactured by Merck). , Enzyme number EC 3.4.23.1) 10 g
Was added and hydrolysis was carried out while stirring at 37 ° C. for 20 hours. The decomposition reaction solution is immediately subjected to an ultrafiltration membrane (Amicon, Y
M10 type, φ76mm) and pass the liquid through
x 50 W x 4 [H ⁺ ] column (φ4.5 x 15 cm)
Added to. After thoroughly washing the column with deionized water,
Elution was performed using 2 L of 2N ammonium hydroxide solution. Ammonia was removed by concentration under reduced pressure to obtain 40 ml of a concentrated liquid. 4 ml of this concentrate was buffered with deionized water in advance.
ephadex G-25 column (φ2.5x150c
m), flow rate 30 ml / hr, fractionation amount 8.6 m
Gel filtration was performed with l. Fractions having a high ACE inhibitory activity, which were collected in a large amount by repeating gel filtration, were collected and lyophilized to obtain a peptide powder. After dissolving 3 g of this peptide in 20 ml of deionized water, SP-S buffered with deionized water in advance was used.
ephadex C-25 (H ⁺ ) column (φ1.5x
(47.2 cm), and perform a concentration gradient method from 1 L of deionized water to 1 L of 3% sodium chloride solution at a flow rate of 3 ml / h.
Chromatography was carried out with r and each fraction amount of 10.0 ml. The result is as shown in FIG. Fractions Nos. 22 to 28 of the ACE inhibitory activity fractions were collected in the above chromatograph and lyophilized to obtain purified tripeptide powder. 20 mg of this purified tripeptide powder was dissolved in 60 μl of deionized water, and then HPLC was performed. As a column, Develosil ODS-5 (4.5 m manufactured by Nomura Chemical Co., Ltd.)
mID × 25 cmL), and the mobile phase is 0.0
A concentration gradient method of 5% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% TFA was performed, and a flow rate was 1.0 ml / min and a detection wavelength was 220 n.
Chromatography was performed at m to obtain a tripeptide having an ACE inhibitory action. The results are shown in FIG. 2, and the elution times of the 23 kinds of tripeptides are shown in Table 1.

The amino acid sequence of the thus-obtained tripeptide having an ACE inhibitory action was determined using a protein sequencer type 447A manufactured by Applied Biosystems. As a result, each of the 23 types of tripeptides It was confirmed to be a tripeptide having a sequence consisting of the L-form amino acid residue represented by. As a result of mass spectrometric analysis of 23 novel tripeptides, it was confirmed that the amino acid sequence and amino acid composition are tripeptides having the amino acid sequence structure represented by the above formula.
The results of this mass spectrum are shown in Table 1.

Production Example 2 [Production of novel tripeptide from soybean] 200 g of soybean
1 L of deionized water was added to and homogenized, and then the pH was adjusted to 2.0 with 1N hydrochloric acid, and pepsin (manufactured by Merck,
Enzyme No. EC3.4.23.1) 10 g was added, and 37
Hydrolysis was performed while stirring at 20 ° C. for 20 hours. The decomposition reaction solution was immediately filtered with an ultrafiltration membrane (Amicon, YM10 type, φ7
6 mm), and the passing liquid is Dowex 50W × 4
It was added to a [H ⁺ ] column (φ4.5 × 15 cm). The column was thoroughly washed with deionized water and then eluted with 2 L of 2N ammonium hydroxide solution. Ammonia was removed by concentration under reduced pressure to obtain 40 ml of a concentrated liquid. This concentrate 4
Sephadex pre-balanced with deionized water
Load G-25 (φ2.5x150cm), flow velocity 30
Gel filtration was performed with ml / hr and each fraction amount of 8.6 ml.
Gel filtration was repeated to collect a large amount of fractions with high ACE inhibitory activity, which were then lyophilized to obtain peptide powder. After dissolving 3 g of this peptide in 20 ml of deionized water, SP-Sephadex C-2 buffered with deionized water in advance was used.
5 [H ⁺ ] column (φ1.5 × 47.2 cm) was loaded, and a concentration gradient method was performed from 1 L of deionized water to 1 L of 3% sodium chloride solution, flow rate 3 ml / hr, each fraction 10.0 m
Chromatography was carried out at l. The result is as shown in FIG.

Fraction Nos. 32 to 3 in the above chromatograph
Fractions of ACE inhibitory activity of 8 were collected and lyophilized to obtain purified tripeptide powder. 20m of this purified tripeptide powder
HPLC was performed after dissolving g in 60 μl of deionized water. As the column, Develos manufactured by Nomura Chemical Co., Ltd.
ilODS-5 (4.5 mmID x 25 cmL) was used, and the mobile phase was 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.
The concentration gradient method of 05% TFA was performed, and the flow rate was 1.0 ml / m.
in, perform chromatograhy at a detection wavelength of 220 nm,
A polypeptide having an ACE inhibitory action was obtained. The results are shown in FIG. 4, and the elution times of 27 tripeptides are shown in Table 2.

The amino acid sequence of the thus-obtained tripeptide having an ACE inhibitory action was determined by using Protein Sequencer 477A type manufactured by Applied Biosystems. As a result, each of the 27 tripeptides It was confirmed to be a tripeptide having a sequence consisting of the L-form amino acid residue represented by. As a result of mass spectrometric analysis of 27 novel tripeptides, it was confirmed that the amino acid sequences and amino acid compositions are tripeptides having the amino acid sequence structure shown by the above formula.
The results of this mass spectrum are shown in Table 2.
The pharmacological effects of the purified fractions of 23 kinds of sardine muscle-derived tripeptide of the present invention and the fractions of 27 kinds of soybean-derived tripeptide of the present invention were confirmed by the following tests.

Test Example 1 [Method for measuring ACE inhibitory activity] 2.5 mU of ACE (manufactured by Sigma, enzyme number EC3.4.5.1), synthetic substrate Hip
puryl-L-his-tidyl-L-leuci
12.5 mM ne (manufactured by Peptide Laboratories) was used for Lieb
The measurement was carried out according to the method of Yamamoto et al., which is an improved method of measuring erman (Nippon Chūshūkai, 18, 297-302 (1989)). That is, the produced hippuric acid was extracted with ethyl acetate and the absorbance was measured at 225 nm. The absorbance of the test solution is Es, and the value when a buffer solution is added instead of the test solution is E
c, The inhibition rate was calculated from the following equation, where Eb is the value when the reaction stop solution was added in advance and the reaction was performed. Inhibition rate (%) = (Ec−Es) / (Ec−Eb) × 10
0 The inhibitory activity IC ₅₀ value of the ACE inhibitor was shown as the concentration (M) of the sample required to inhibit the enzyme activity of ACE by 50% (inhibition rate). Novel 23 derived from sardine muscle according to the present invention
Inhibitory Activity of Some Tripeptides on Bovine Lung Serum ACE
(IC ₅₀ ) is as shown in Table 1. Further, 27 novel tripeptides derived from soybean according to the present invention, bovine lung serum A
The inhibitory activity (IC ₅₀ ) against CE is shown in Table 2.

Test Example 2 [Effect of hypotension upon administration of novel tripeptide to rat] Experimental Material The purified tripeptide powder obtained in the above Production Examples 1 and 2. That is, a fraction consisting of 23 kinds of sardine muscle-derived tripeptide (SP-1 fraction) and a fraction consisting of 27 kinds of soybean-derived tripeptide (SP-1 fraction) were used. II. Experimental method As an experimental animal, 15-week-old male spontaneously hypertensive rat (SHR) was purchased from Charles River Japan, Inc., and after preliminarily breeding for 1 week, an animal 6 having a systolic blood pressure of 160 mmHg or more (body weight 280 to 330 g) 6 The animals were used as one group. Rats have room temperature of 23 ± 25 ° C, humidity of 55 ± 10% and 12
In a breeding room adjusted to the time light and dark (lights from 6 am to 6 am), one animal was housed in each individual cage for stainless steel rats and bred. The feed is M from Oriental Yeast Factory Co., Ltd.
F powdered feed and self-pumping (conforming to tap water quality standard) were freely taken as drinking water. 4 groups of rats (6 per group)
As a control, distilled water was forcibly orally administered to the first group at a rate of 0.5 ml per 100 g of body weight, and to the second group, a dose of 1.0 g / kg of tripeptide powder was prepared with distilled water. Gavage was orally administered at a rate of 0.5 ml per 100 g of body weight, and tripeptide powder was 2.0 g / k for the third group.
g, a dose of 4.0 g / kg of tripeptide powder was orally administered to the fourth group in the same manner as in the second group.

Blood pressure was measured using a non-invasive tail artery blood pressure measuring device (PS-100, manufactured by Riken Development Co., Ltd.) tail-
By cuff method, before administration, 30 minutes, 1 hour, 2 after administration
Blood pressure and heart rate were measured at 4 hours and 6 hours. Blood pressure was measured continuously 3 times, and when the difference between the highest value and the lowest value was within 10 mmHg, the average blood pressure value of the 3 times was calculated. If the difference is 11 mmHg or more, measure twice more,
Except for the highest value and the lowest value, the mean blood pressure value was calculated three times.
In addition, the average heart rate was obtained using the measurement value when the average blood pressure value was calculated. The results of the effects on blood pressure and heart rate after single oral administration of sardine muscle-derived tripeptide fraction (SP-1 fraction) 300,600 and 1,200 mg / kg using SHR were as follows: As shown in Table 3 and FIG. Also, using SHR, soybean-derived tripeptide fraction (SP-1 fraction) 300, 600 and 1,
The results of the effects on blood pressure and heart rate after a single oral administration of 200 mg / kg are shown in Table 4 and FIG. As a result of the above test, the fraction consisting of 23 kinds of sardine muscle-derived tripeptides according to the present invention and the fraction consisting of 27 kinds of soybean-derived tripeptides have ACE inhibitory activity and have a significant antihypertensive effect in vivo. It was confirmed that Therefore, 23 kinds of sardine muscle-derived tripeptides and 27 kinds of soybean-derived tripeptides according to the present invention are useful as therapeutic or preventive agents for hypertension. In addition, 23 kinds of sardine muscle-derived tripeptides and 27 kinds of soybean-derived tripeptides according to the present invention can also be adopted in the structure in a peptide whose amino acid sequence is a partial structure.

[0019]

[Table 1] Elution time, amino acid sequence, molecular weight and inhibitory activity of sardine muscle-derived tripeptide by HPLC.

[0020]

[Table 2] Elution time, amino acid sequence, molecular weight and inhibitory activity of soybean-derived tripeptide in HPLC.

[0021]

[Table 3] Changes in blood pressure of SHR after administration of tripeptide fraction derived from sardine muscle

[0022]

[Table 4] Changes in blood pressure of SHR in soybean-derived tripeptide fractionation

[0023]

[Brief description of drawings]

FIG. 1 shows a sardine muscle-derived tripeptide according to the present invention,
SP-Sephad-ex C-25 in Production Example 1
It is a figure which shows the result of isolation and purification of the ACE inhibitory peptide by (H ⁺ ) column chromatography.

FIG. 2 SP-Sephadex C-25 (H ⁺ ) of Production Example 1 of the soybean-derived tripeptide according to the present invention.
It is a figure which shows the result of the separation purification of the ACE inhibitory peptide by column chromatography.

FIG. 3 shows a sardine muscle-derived tripeptide according to the present invention,
FIG. 6 is a diagram showing the results of inconvenient purification of an ACE-inhibiting peptide by reverse phase HPLC in Production Example 1.

FIG. 4 is a diagram showing the results of separation and purification of a soybean-derived tripeptide according to the present invention by reverse phase HPLC of an ACE-inhibiting peptide in Production Example 1.

FIG. 5: 23 kinds of tripeptide fractions obtained in Production Example 1 of sardine muscle-derived tripeptide according to the present invention (SP-1
FIG. 3 is a diagram showing a change in blood pressure value over time when a fraction) is administered to SHR.

FIG. 6 Production Example 2 of soybean-derived tripeptide according to the present invention
27 kinds of tripeptide fractions obtained in (SP-1 fraction)
FIG. 3 is a graph showing changes in blood pressure values over time when is administered to SHR.

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Claims (6)

[Claims] 1. 23 novel tripeptides having a peptide structure according to the L-amino acid sequence represented by: 2. A product obtained by treating sardine muscle with a proteolytic enzyme is filtered, and the components of the filtrate component that have passed through the semipermeable membrane are sequentially filtered with a strong acid cation exchange resin, gel filtration, A fraction containing a component having angiotensin converting enzyme inhibitory activity is obtained from each fraction obtained by fractionation by ion-exchange gel filtration and reverse phase high performance liquid chromatography. The new 2
A method for producing three types of tripeptides. 3. An antihypertensive agent comprising one or more tripeptides selected from the novel 23 kinds of tripeptides of claim 1 as an active ingredient. 4. 27 novel tripeptides having a peptide structure according to the L-amino acid sequence represented by: 5. A product obtained by treating soybean with a proteolytic enzyme is filtered, and the components of the filtered component that have passed through the semipermeable membrane are sequentially filtered with a strong acid cation exchange resin, gel filtration,
A fraction containing a component having angiotensin converting enzyme inhibitory activity is obtained from each fraction obtained by fractionation by ion-exchange gel filtration and reverse phase high performance liquid chromatography. Of 27 novel tripeptides of 6. An antihypertensive agent comprising, as an active ingredient, one or more tripeptides selected from the 27 novel tripeptides of claim 4.