JP2007182414A - New dried bonito peptide, l-valyl-l-proline and antihypertensive agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new dried bonito peptide and L-valyl-L-proline having angiotensin converting enzyme inhibitory effect and hypotensive effect from a liquid obtained by degrading dried bonito with protease: Molsin F. <P>SOLUTION: The dipeptide obtained by carrying out degradation treatment of dried bonito with protease: Molsin F and having a new angiotensin converting enzyme inhibitory effect is L-valyl-L-proline. The new dried bonito peptide has hypotensive effect in the living bodies and extremely low toxicity. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a novel bonito peptide having a dipeptide structure represented by L-valyl-L-proline, which has utility as a pharmaceutical product, and an antihypertensive agent comprising the novel bonito peptide as an active ingredient.

Food-derived peptides have advantages as angiotensin converting enzyme inhibitors and antihypertensive agents.
JP 06-256387 JP-A-06-340692 JP 07-188282 A JP 07-188283 A JP 08-225593 A JP 09-059296 A Japanese Patent Laid-Open No. 10-033691 JP 11-335393 A JP-A-11-343298 JP 2001-064299 A JP 2001-106698 A JP 2001-106699 A JP 2001-106700 A JP 2003-128695 A S. H. Ferreia et al. : Biochemistry, 9, 3583 (1970) G. Oshima et al. : Biochim. Biophs. Acta, 566, 128 (1979) S. Maruyama et al. : Agric. Biol. Chem. 46, 1393 (1983) Setsuko Yamamoto, etc .: Journal of the Japan Breast Society, 18, 297-302 (1989)

It is well known that the renin-angiotensin system plays an important role in the regulation of biological water, electrolytes and blood. This renin-angiotensin system contains an angiotensin converting enzyme (hereinafter abbreviated as ACE), and angiotensin I is converted to angiotensin II by ACE. Angiotensin II is a powerful vasopressor that works not only in blood vessels and adrenal cortex but also in the central nervous system and peripheral nervous system to promote an increase in blood pressure. ACE also has the action of degrading and inactivating bradykinin, which is a hypotensive substance in vivo, and is involved in the pressor system. Therefore, it is possible to lower blood pressure by inhibiting the activity of ACE, and this is clinically effective for the prevention and treatment of hypertension. Since captoryl, a proline derivative, was synthesized for this purpose and its antihypertensive activity was confirmed, various ACE inhibitors have been actively studied based on the structural study of captolyl. Recently, enalabril maleate, alacebryl, etc. These substances are being used in clinical settings one after another. Currently, ACE inhibitors are widely used for hypertension regardless of whether it is essential hypertension or symptomatic hypertension, and whether it is mild or severe. And have been found to have many excellent points. On the other hand, as the mechanism of action of ACE inhibitors, it is considered that aldosterone and vasopressin secretion are suppressed by suppressing production of angiotensin II, and that sodium and water excretion is promoted by releasing renal artery contraction. Furthermore, for ACE inhibitors, it is believed that it suppresses inactivation of the calicrene-kinin system and further promotes peripheral vasodilation and excretion of sodium and water by activating the prostaglandin system, It is expected to be an appropriate therapeutic agent in breaking the vicious circle of heart failure. By the way, as an ACE inhibitor, in addition to the above-mentioned synthetic product, a snake venom-derived bradykinin enhancing factor (C-terminal is Pro) [Non-patent document 1] as a substance derived from a natural product or a natural product, derived from a collagenase digest of gelatin Beginning with six types of peptides (all C-terminal is Ala-Hyp) [Non-patent document 2], peptides derived from tryptic digest of bovine casein (C-terminal is Gly-Lys) [Non-patent document 3], etc. 5 types of hexapeptides derived from sardine muscles (all are 2nd or 3rd from the C-terminal, Pro is N-terminal, Leu is N-terminal), tetrapeptide derived from laver (N-terminal is Pro), pentagon derived from ginseng There are numerous patents [Patent Documents 1 to 14] such as peptides, chlorella-derived pentapeptides and wakame-derived tetrapeptides, all of which are ACE inhibitors. It is disclosed that can be a fine antihypertensive agent. As described above, many proposals have been made on blood pressure-lowering peptides derived from food proteins as ACE inhibitors. Among the peptides derived from bonito, ACE inhibition of dipeptides having a regular amino acid sequence. The effects (in vitro pharmacological effects) and antihypertensive effects by oral administration (in vivo pharmacological effects) are unknown, and there is no report that development as a pharmaceutical product has progressed since its discovery ( Each symbol represented is in accordance with the convention used in amino acid chemistry).

As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that the novel bonito peptide according to the present invention obtained from bonito has a blood pressure lowering action and has completed the present invention. That is, a substance having a pharmacological action was searched from the decomposition solution of bonito proteolytic enzyme, and the novel bonito peptide was found to have a strong angiotensin converting enzyme inhibitory action. And, as a result of intensive research for practical application of this novel bonito peptide as a pharmaceutical, this novel bonito peptide has a blood pressure lowering effect and is useful as an angiotensin converting enzyme inhibitor derived from natural products. I found. The present invention is based on such knowledge. The novel bonito peptide according to the present invention has a dipeptide structure represented by L-valyl-L-proline, and is white powder at room temperature.

The novel bonito peptide according to the present invention can be chemically synthesized or separated and purified from the bonito proteolytic enzyme degradation solution. In the case of chemically synthesizing the novel bonito peptide according to the present invention, it can be carried out by a usual synthesis method such as a liquid phase method or a solid phase method. The L-form amino acids corresponding to the amino acid residues are sequentially bound to the body from the carboxyl terminal side (C-terminal) of the bonito peptide by peptide bonds. The synthetic dipeptide thus obtained is cleaved from the porous solid support using trifluoromethanesulfonic acid, hydrogen fluoride, etc., and then the amino acid side chain protecting group is removed, and the reverse phase system is removed. And purified by a conventional method using high performance liquid chromatography (hereinafter abbreviated as HPLC).

As described above, the novel bonito peptide according to the present invention can be separated and purified from the decomposition solution of bonito proteolytic enzyme. In that case, for example, it can be carried out as follows. Hydrolysis is performed using bonito containing the novel bonito peptide according to the present invention. The hydrolysis method is performed according to a conventional method. For example, when hydrolyzing with a proteolytic enzyme such as morsine F, etc., if necessary, further hydrolyze the bonito homogenate, then warm to the optimal temperature of the enzyme, adjust the pH to the optimal value, Add and incubate. Next, after neutralization as necessary, the enzyme is deactivated to obtain a hydrolyzed solution. The hydrolyzate is filtered using filter paper and / or celite to remove insoluble components, and the obtained filtrate is filtered using a semipermeable membrane such as cellophane to form a suitable solvent (for example, water, tris-hydrochloric acid). Dialyze sufficiently in a buffer solution or neutral buffer solution of a phosphate buffer solution, and a solution containing a component that has passed through a semipermeable membrane with a component in the filtrate is a strongly acidic cation exchange resin (for example, Dow Chemical). And then a fraction containing an angiotensin converting enzyme (hereinafter abbreviated as ACE) inhibitory activity is obtained from the adsorbed elution fraction. The obtained ACE inhibitory activity fraction was subjected to gel filtration (for example, Sephadex G-25 manufactured by Pharmacia, Bio-Gel P-6 manufactured by Bio-Rad, etc.). The obtained ACE inhibitory activity fraction is fractionated by cation exchange gel filtration (for example, SP-Sephadex C-25 manufactured by Pharmacia, SE-Cellulose manufactured by Seikagaku Corporation), and the like. Isolation and purification can be carried out by separating the obtained ACE inhibitory activity fraction by reverse phase HPLC.

As the seafood section used in the method for producing the novel bonito peptide according to the present invention, any seafood section may be used as long as the object of the present invention can be achieved, but bonito is preferably used. The novel bonito peptide obtained as described above does not cause antibody production and does not cause anaphylactic shock when repeatedly administered intravenously. In addition, since this novel bonito peptide has a sequence structure of only L-amino acid and is gradually decomposed by protease in vivo after administration, the toxicity is extremely low and the safety is extremely high (LD ₅₀ > 5000 mg / kg). Rat oral administration). This novel bonito peptide can be prepared into injections, tablets, capsules, granules, powders, etc. using commonly used additives such as excipients. The administration method usually includes injection into a mammal having ACE (for example, human, dog, rat, etc.) or oral administration. The dosage is, for example, 0.01 to 10 mg of this novel bonito peptide per animal body weight. The number of administration is usually about 1 to 4 times a day, but can be appropriately adjusted depending on the administration route.

The types of excipients, binders, and lubricants used in the various preparations are not particularly limited, and those used for ordinary injections, powders, granules, tablets, or capsules can be used. Examples of additives used in tablets, capsules, granules, and powders include the following. As excipients, sugars such as crystalline cellulose, sugar alcohols such as mannitol, starches, anhydrous calcium phosphate, etc .; starches, hydroxypropyl methylcellulose, etc. as binders; Examples of the lubricant include stearic acid and its salts, talc, and waxes. In preparation of the preparation, flavoring agents such as menthol, citric acid and salts thereof, and fragrance can be used as necessary. A sterile composition for injection is prepared by dissolving or suspending a novel bonito peptide in water for injection, physiological saline, a sugar alcohol injection solution such as xylitol or mannitol, or a glycol such as propylene glycol or polyethylene glycol by a conventional method. It can be an injection. At this time, a buffer solution, a preservative, an antioxidant and the like can be added as necessary. The preparation containing the novel bonito peptide is in the form of a lyophilized product or a dry powder, and can be used by dissolving it in an ordinary solubilizing agent such as water or physiological saline at the time of use.

The novel bonito peptide 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, prevention of hypertension such as essential hypertension, renal hypertension, adrenal hypertension, therapeutic agent, normalization of organ circulation and improvement of long-term prognosis (life extension effect) for these congestive heart failure, treatment of heart failure Useful as an agent.

BEST MODE FOR CARRYING OUT THE INVENTION

The novel bonito peptide according to the present invention has a dipeptide structure represented by L-valyl-L-proline, and is white powder at room temperature. EXAMPLES Examples and test examples will be described below as examples, and the present invention will be described in more detail. However, the present invention is not limited to these examples.

Production Example 1
After 7.16 g of a 7-day dried product (coarse portion) produced by the ventilation roasting method was cut finely, 1 L of deionized water was added and homogenized. Morsin F (manufactured by Shengshin Pharmaceutical Co., Ltd.) (34.8 g) was added to the obtained bonito homogenate, adjusted to pH 3.0, and incubated at 37 ° C. for 20 hours. The bonito homogenate molsin decomposition solution thus prepared was ultrafiltered using a Diaflow membrane (Amicon, YM-10 type membrane, molecular weight cut off 10,000). The obtained filtrate was chromatographed using a column packed with Dowex 50W × 4 (H ⁺ ). The extract was washed with deionized water, elution was performed with 2N-NH ₄ OH, and the eluate was concentrated. This concentrated solution was chromatographed using a Sephadex G-25 column to fractionate the peptide fraction (fraction numbers 24 to 35). The column chromatograph is shown in FIG. The peptide fraction was concentrated to obtain a bonito-derived peptide solution (crude peptide powder). Furthermore, this peptide solution (1.5 g as a crude peptide powder) was chromatographed with an SP-Sephadex C-25 (H ⁺ ) column to obtain SP-1 fractions (fraction numbers 13 to 31) as the peptide fractions, The SP-2 fraction (fraction numbers 32-53) and SP-3 fraction (fraction numbers 54-70) were separated. The column chromatograph is shown in FIG. Each of these fractions was lyophilized to obtain 0.72 g of SP-1 fraction, 0.32 g of SP-2 fraction, and 0.28 g of SP-3 fraction as peptide fraction derived from bonito (purified peptide powder). It was. Of the purified peptide powder fractionated in this way, the purified peptide powder of the SP-3 fraction with high ACE inhibitory activity was dissolved in deionized water (7 mg / 25 μL) and then subjected to HPLC. The conditions were as follows: Develosil ODS-5 (¢ 4.6 mm ID × 25 cmL) manufactured by Nomura Chemical Co., Ltd. was used as the column, and 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05 as the mobile phase. Chromatography was performed at a flow rate of 1.0 mL / min and a detection wavelength of 220 nm by a concentration gradient method using% TFA to obtain a peptide fragment having a strong ACE inhibitory action at an elution time of 45.7 minutes. The result is as shown in FIG. The amino acid sequence of the thus obtained peptide having ACE inhibitory action was determined using a protein sequencer type 477A manufactured by Applied Biosystems (ABI). As a result, it was confirmed to be a bonito peptide having a dipeptide structure represented by L-valyl-L-proline. The property at room temperature is a white powder.
In addition, when the bonito peptide according to the present invention is formulated as an ACE inhibitor, for example, into a tablet, it may be processed according to a conventional method, for example, as follows: (1) 13 g of peptide, (2) 87 g of lactose, ( 3) 29 g of corn starch, (4) 1 g of magnesium stearate, first (1), (2) and 17 g of corn starch are mixed and granulated with a paste made from 7 g of corn starch, and 5 g of corn starch and (4) is added, and the resulting mixture is compressed with a compression tablet machine to produce 1000 tablets.

Production Example 2
This example is an example of production by a synthesis method.
Synthesis method of L-vanyl-L-proline: The dipeptide was synthesized by a solid phase method using an automatic peptide synthesizer type 430A manufactured by Applied Biosystems. As the solid support, a resin obtained by chloromethylating a styrene divinylbenzene copolymer (polystyrene resin) was used. First, according to the amino acid sequence of the said dipeptide, it reacted with the chloromethyl resin from the proline of the C terminal side as usual, and obtained the peptide bond resin. As the amino acid at this time, a t-Boc amino acid protected with a t-butoxycarbonyl (hereinafter abbreviated as t-Boc) group was used. Next, this peptide-bonded resin is suspended in a mixed solution composed of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, added with trifluoroacetic acid (hereinafter abbreviated as TFA) under ice cooling, and further stirred for 10 minutes. did. Trifluoromethanesulfonic acid was added dropwise to the mixture, and the mixture was stirred at room temperature for 30 minutes. Then, anhydrous ether was added to precipitate the product, and the precipitate was washed several times with anhydrous ether. And dried. The crude synthetic peptide thus obtained was dissolved in distilled water and then purified by HPLC using a reverse phase system column C ₁₈ (5 μm). (A) 0.1% TFA-containing distilled water and (B) 0.1% TFA-containing acetonitrile solution were used as the mobile phase, and (A) liquid was flowed at a flow rate of 1 by a concentration gradient method of 87% → 68% over 20 minutes. Chromatography was performed at 6 mL / min. The elution fraction detected at an ultraviolet wavelength of 214 nm and exhibiting the maximum absorption was collected and lyophilized to obtain the target synthetic dipeptide.

This synthetic dipeptide was confirmed to be a knot peptide having a dipeptide structure represented by L-valyl-L-proline in the amino acid sequence by mass spectrum analysis, amino acid analysis and amino acid sequencer. The pharmacological effect of the dipeptide according to the present invention obtained by synthesis, that is, a novel bonito peptide, was confirmed by the following tests.

Test example 1
Angiotensin converting enzyme inhibitory activity measurement method: ACE (manufactured by Sigma, enzyme number EC 3.4.15.1) 2.5 mU, synthetic substrate: Hipril-L-histidyl-L-leucine (manufactured by Peptide Institute) 12.5 mM The measurement was performed according to the method of Yamamoto et al. That is, the produced hippuric acid was extracted with ethyl acetate and measured by absorbance at 225 nm. Inhibition rate (%) from the following equation, where Ab is the absorbance in the test solution, Ec is the value when the buffer solution is added instead of the test solution, and Eb is the value when the reaction stop solution is added in advance to react. Asked.
Inhibition rate (%) = (Ec−Es) / (Ec−Eb) × 100
The inhibitory activity IC ₅₀ value of the ACE inhibitor was expressed as the concentration (M) of the sample required to inhibit the enzyme activity of ACE by 50%. The inhibitory activity (IC ₅₀ value) of dipeptide: L-valyl-L-proline of the present invention against angiotensin converting enzyme in bovine lung serum was as high as 1.13 μM.

又、本発明に係る合成ジペプチド；Ｌ−バリル−Ｌ−プロリン１０ｍｇ／ｋｇをＳＨＲに９週間（Ｗ）まで経口投与した時の各血圧値（ｍｍＨｇ）についての結果は、表２に示すとおりである。

これらの試験の結果、本発明係るジペプチドすなわち新規なかつお節ペプチドは、アンジオテンシン変換酵素阻害活性を有し、ｉｎ ｖｉｖｏ（生体内）においても有意な血圧降下作用を示すことが確認された。従って、本発明係るジペプチドすなわち新規なかつお節ペプチドは高血圧症の治療又は予防薬として有用である。尚、本発明係るジペプチドすなわちＬ−バリル−Ｌ−プロリンは、構造的にそのアミノ酸配列を部分構造とするペプチドにおいて、構造中に採用することもできる。Test example 2
Antihypertensive effect when administered to spontaneously hypertensive rats: Experimental animals purchased 15-week old male spontaneously hypertensive rats (hereinafter abbreviated as SHR) from Charles River, Japan, and after 1 week of pre-breeding, contraction period A group of 3 animals having a blood pressure of 160 mmHg or more (weight 280 to 330 g) was used. Rats were housed individually in stainless steel rat individual gauges in a breeding room adjusted to room temperature 23 ± 2 ° C., humidity 55 ± 10%, and 12 hours light / dark (lighting from 6 am to 6 pm). . The feed was MF powder feed manufactured by Oriental Yeast Co., Ltd., and the drinking water was ingested by private pumping (conforming to tap water quality standards). Blood pressure was determined by tail-cuff method using a non-invasive tail artery blood pressure measuring device (manufactured by Riken Development Co., Ltd., model PS-100), with 0 hours before administration, 27 hours after administration, and 9 weeks after administration. In this period, the measurement of the systolic blood pressure (mmHg, upper value), the average blood pressure (mmHg) and the diastolic blood pressure (mmHg, lower value) of the SHR tail artery was performed 5 times at regular time intervals, The highest value and the lowest value were rejected, and the average value of three times was taken as the measured value at each time. The results for each blood pressure value (mmHg) when 10 mg / kg L-valyl-L-proline according to the present invention was orally administered to SHR for up to 27 hours (h) are as shown in Table 1.

Moreover, the result about each blood-pressure value (mmHg) when the synthetic dipeptide which concerns on this invention; 10 mg / kg of L-valyl-L-proline is orally administered to SHR for 9 weeks (W) is as showing in Table 2. is there.

As a result of these tests, it was confirmed that the dipeptide according to the present invention, that is, a novel bonito peptide has an angiotensin converting enzyme inhibitory activity and exhibits a significant blood pressure lowering effect in vivo (in vivo). Therefore, the dipeptide according to the present invention, that is, a novel bonito peptide is useful as a therapeutic or prophylactic agent for hypertension. In addition, the dipeptide which concerns on this invention, ie, L-valyl-L-proline, can also be employ | adopted in a structure in the peptide which has the amino acid sequence as a partial structure structurally.

It is a figure which shows the result of the separation and refinement | purification of the bonito derived peptide by Sephadex G-25 column chromatography in the manufacture example 1 of the molsin decomposition solution of the bonito concerning this invention. The markers in the figure are insulin having a molecular weight of 6,000, insulin B chain having a molecular weight of 3,500, insulin A chain having a molecular weight of 2,550, bacitracin having a molecular weight of 1,450, and glycine having a molecular weight of 75. It is a figure which shows the result of the isolation | separation purification of the bonito-derived peptide by SP-Sephadex C-25 (H ^<+> ) column chromatography in the manufacture example 1 of the bonito peptide concerning this invention. It is a figure which shows the result of isolation | separation and the refinement | purification of the fragment of the peptide which has the ACE inhibitory effect by reverse phase HPLC in the manufacture example 1 of the bonito peptide concerning this invention.

Claims (2)

A novel bonito peptide having a dipeptide structure represented by the following formula: L-valyl-L-proline. A blood pressure-lowering agent comprising a novel bonito peptide having a dipeptide structure represented by the following formula: L-valyl-L-proline as an active ingredient.

Images