JP2003246795A - Novel undaria peptide, l-isoleucyl-l-tyrosine, and antihypertensive agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel undaria peptide, i.e., L-isoleucyl-L-tyrosine, which has an angiotensin converting enzyme-inhibiting action and an antihypertensive action and is obtained from a solution prepared by decomposing undaria with proteases S AMANO (R) and PROLEATHER (R) FG. <P>SOLUTION: The novel undaria peptide, i.e., a dipeptide, obtained by decomposing undaria with protease and the like and having a novel angiotensin converting enzyme-inhibiting action is L-isoleucyl-L-tyrosine and has an in-vivo antihypertensive action and a very low toxicity. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel wakame peptide having a dipeptide structure represented by L-isoleucyl-L-tyrosine, which is useful as a medicine, and an antihypertensive agent containing the novel wakame peptide as an active ingredient. .

[0002]

2. Description of the Related Art In recent years,
The present inventors have reported pharmacologically active peptides such as angiotensin converting enzyme (ACE) inhibitory peptides during enzymatic degradation of seaweed-derived food proteins. That is, as an ACE inhibitory peptide derived from seaweed protein, Ile-
Tyr; Met-Lys-Tyr; Ala-Lys-T
yr-Ser-Tyr; Leu-Arg-Tyr et al., “Marine Biotechnology, vol. 6, page 163, 1998.
“Year” represents Gly-Lys-Tyr; Ser-Val-Ty as the ACE inhibitory peptide derived from the Hijiki protein.
r; Ser-Lys-Thr-Tyr et al., "Journal of the Fisheries Society of Japan, Vol. 64, page 862, 1998" has been reported. As microalgae-derived, Ile-Val as an ACE inhibitory peptide derived from Chlorella spirulina protein
-Val-Glu; Ala-Phe-Leu; Phe-
Ala-Leu, Ala-Glu-Leu, Val-V
al-Pro-Ala, Ile-Ala-Glu; Il
e-Ala-Pro-Gly, Val-Ala-Phe
"Marine Biotechnology, Volume 3, 305 pages, 20"
'01 ”has been reported. Moreover, from the garlic extract, Ser-Tyr; Gly-Tyr; Phe-Ty
r; Asn-Tyr; Ser-Phe; Gly-Ph
e; Asn-Phe et al., “Journal Nutritional Bichemistry, Volume 9, 415, 1998”
Has been reported. Furthermore, as an ACE inhibitory peptide derived from wakame protein, Ala-Ile-Tyr-
Lys; Tyr-Lys-Tyr-Tyr; Lys-P
he-Tyr-Gly; Tyr-Asn-Lys-Le
"Journal Nutritional Bichemistry, Vol. 11, p. 450, 2000" has been reported. By the way, it is well known that the renin-angiotensin system plays an important role in regulating water / electrolytes and blood in the living body. Angiotensin converting enzyme (ACE) exists in this renin-angiotensin system, and angiotensin I is converted to angiotensin II by ACE. Angiotensin II is a powerful pressor substance that acts on not only blood vessels and adrenal cortex but also central nervous system and peripheral nervous system to promote an increase in blood pressure. Further, ACE has a function of decomposing and inactivating bradykinin which is an antihypertensive substance in vivo, and is involved in the pressor system. Therefore, AC
It is possible to lower blood pressure by inhibiting the activity of E, which is clinically possible to prevent hypertension,
It is believed to be effective in treatment. For this purpose, caprolyl, a proline derivative, was synthesized, and since its hypotensive action was confirmed, various ACE inhibitors have been actively researched on the basis of the structure study of captolyl, and recently, enarabril maleate, aracebryl, etc. Substances are being offered to clinical sites one after another. Currently, ACE inhibitors are widely used regardless of whether they are essential hypertension or symptomatic hypertension, mild or severe, and they are added to the first-line treatment of hypertension. It has been found to have points. On the other hand, as the mechanism of action of the ACE inhibitor, suppression of the secretion of aldosterone and vasopressin by suppressing the production of angiotensin II, and promotion of sodium and water excretion by releasing the contraction of renal arteries are considered.
Furthermore, regarding the ACE inhibitor, it is considered that it suppresses the inactivation of the calyculene-quinine system and activates the prostaglandin system to further promote peripheral vasodilation and excretion of sodium and water, It is expected as a purposeful therapeutic drug for breaking the vicious circle of heart failure. A
As the CE inhibitory substance, in addition to the above synthetic products, a natural product or a substance derived from a natural product, a bradykinin-enhancing factor derived from snake venom (C-terminal is Pro) [S. H. Ferria et
al: Biochemistry, 9, 3583 (1
970)], 6 types of peptides derived from collagenase digestion products of gelatin (all have C-terminal Ala-Hyp).
[G. Oshima et al: Biochim. Bi
ophs. Acta, 566, 128 (1979)],
A peptide derived from a tryptic digest of bovine casein (Gly-Lys at the C-terminus) [S. Maruyama et a
l. : Agric. Biol. Chem. , 46, 13
93 (1983)] and the like, and five hexapeutides derived from the sardine muscle of the present inventors (all are the second or third from the C terminus as Pro and the N terminus as Leu) [Patent No. 20]
No. 46483], a nori protein-derived tetrapeptide (Pro-Gly-Val-Ala) [Patent No. 2678].
180], pentapeptide derived from ginseng protein (Ile-Gly-Pro-Ala-Gly) [Patent No. 2920829], pentapeptide derived from chlorella protein (Val-Val-Pro-Pro-Ala).
And tetrapeptides derived from three types of wakame proteins (T
yr-Asn-Lys-Leu, Tyr-Lys-Ty
r-Tyr, Ala-Ile-Tyr-Lys) [Patent No. 3108920] and the like are mentioned, and it is disclosed that any of them can be an ACE inhibitor. As described above, many proposals concerning the wakame peptide derived from the wakame protein as the ACE inhibitor have been made by the present inventor, including many Japanese Patent Applications No. 2001-113295 and No. 2001-11.
8914, Japanese Patent Application No. 2001-228754, Japanese Patent Application 20
No. 01-355368 ”, the ACE inhibitory effect of dipeptide having a regular amino acid sequence among wakame peptides (pharmacological effect in vitro) and hypotensive effect by oral administration (pharmacological effect in vivo) It is unknown, and there is no report that it is still being developed as a drug since its discovery. (In the formula, each symbol representing an amino acid residue is based on a conventional notation in amino acid chemistry.)

[0003]

[Means for Solving the Problems] The present inventor searched for a substance having a pharmacological action from a degradation solution of a proteolytic enzyme of wakame seaweed belonging to the seaweed species of the brown seaweed Laminariales, and found that the novel wakame peptide has strong angiotensin. It was found that it has a converting enzyme inhibitory action. And
We have earnestly conducted research to put the novel wakame peptide into practical use as a medicine. As a result, they found that this novel wakame peptide has a hypotensive action and is useful as an angiotensin converting enzyme inhibitor derived from a natural product. The present invention is based on such findings. The novel wakame peptide according to the present invention has a dipeptide structure represented by L-isoleucyl-L-tyrosine, and is a white powder at room temperature.

The novel wakame peptide according to the present invention can be chemically synthesized or separated and purified from a decomposing solution of wakame proteolytic enzyme.
In the case of chemically synthesizing the novel wakame peptide according to the present invention, it can be carried out by an ordinary peptide synthesis method such as a liquid phase method or a solid phase method. To phase support L-isoleucyl-
L-tyrosine corresponding to the carboxyl-terminal (C-terminal) amino acid of L-tyrosine may be sequentially linked by peptide bonds. The synthetic dipeptide thus obtained is trifluoromethanesulfonic acid,
After cleaving from the solid support of the poromeric property using hydrogen fluoride, etc., the protecting group of the amino acid side chain is removed, and ordinary high-performance liquid chromatography (HPLC) using a reversed-phase column is used. It can be purified by a method.

As described above, the novel wakame peptide according to the present invention can be separated and purified from the degradation solution of the seaweed proteolytic enzyme. In that case, for example, it can be carried out as follows. . The protein portion of wakame containing the novel wakame peptide described above is used for hydrolysis. Hydrolysis is performed according to a conventional method. For example, when hydrolyzing with proteases such as Protease S Amano and Prolezer FG-1, wakame is further hydrolyzed if necessary and then heated to the optimum temperature of the enzyme to adjust the pH to the optimum value ( The pH is adjusted to 8.0 and pH 10.0) and the enzyme is added and incubated. Then, after neutralizing as necessary, the enzyme is deactivated to obtain a hydrolyzed solution. The hydrolyzate is filtered with filter paper and / or Celite to remove insoluble components, and the obtained filtrate is filtered with a semipermeable membrane such as cellophane to obtain a suitable solvent (eg,
A solution containing the components that have passed through the semipermeable membrane among the components in the filtrate after being sufficiently dialyzed in water, Tris-hydrochloric acid buffer solution, neutral buffer solution of phosphate buffer solution, etc. is strongly acidic cation exchange resin. (For example, Dowex 50W manufactured by Dow Chemical Co., Ltd.),
From the adsorbed and eluted fraction, angiotensin converting enzyme (AC
E) A fraction containing a component having inhibitory activity was obtained, and the obtained ACE inhibitory activity fraction was fractionated by gel filtration (for example, Sephadex G-25 manufactured by Pharmacia) to obtain the obtained ACE inhibitory activity. The fraction was subjected to cation exchange gel filtration (for example, SP-Sephade manufactured by Pharmacia).
x C-25 etc.), and the ACE inhibitory activity fractions obtained by these chromatographic purifications are further fractionated by reverse phase high performance liquid chromatography (HPLC).

As the brown algae used in the method for producing the novel wakame peptide according to the present invention, any brown algae may be used as long as the object of the present invention can be achieved, but it is preferable to use wakame. The novel wakame peptide according to the present invention obtained as described above does not induce antibody production or anaphylactic shock when repeatedly administered intravenously. Further, the novel wakame peptide according to the present invention has a sequence structure of only L-amino acids, and is gradually degraded by in vivo protease after administration, and therefore has extremely low toxicity and extremely high safety (LD
₅₀ > 5000 mg / kg; rat oral administration). The novel wakame peptide according to the present invention can be prepared into injections, tablets, capsules, granules, powders and the like by using additives such as commonly used excipients. 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
The amount of this wakame peptide is 0.01 to 10 mg per kg. The number of administrations is usually about 1 to 4 times a day,
It can be appropriately prepared depending on the administration route.

The kinds of excipients, binders and lubricants used in the above-mentioned various preparations are not particularly limited, and those used for ordinary injections, powders, granules, tablets or capsules should be used. You can The following may be mentioned as additives used in tablets, capsules, granules and powders. Excipients include sugars such as crystalline cellulose, sugar alcohols such as mannitol, starches, anhydrous calcium phosphate and the like; binders such as starch and hydroxypropylmethyl cellulose; disintegrators such as carboxymethyl cellulose and potassium salts thereof; Examples of lubricants include stearic acid and its salts, talc, and waxes. In preparation of the preparation, menthol, citric acid and its salts, and flavoring agents such as fragrances can be used if necessary. A sterile composition for injection is prepared by dissolving or suspending the novel peptide of the present invention in water for injection, physiological saline, a sugar alcohol injection such as xylitol or mannitol, or a glycol such as propylene glycol or polyethylene glycol by a conventional method. It can be made turbid to prepare an injection. At this time, a buffer solution, a preservative, an antioxidant and the like can be added as required. The preparation containing the novel wakame peptide according to the present invention may be in the form of a lyophilized product or a dry powder, and may be dissolved in an ordinary solubilizer such as water or physiological saline before use.

The novel wakame 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, a prophylactic / therapeutic agent for hypertension such as essential hypertension, renal hypertension, and adrenal hypertension, which has the effects of normalizing the organ circulation and improving the long-term prognosis (prolonging survival) for congestive heart failure, and treating heart failure. It is useful as an agent.

[0009]

EXAMPLES Hereinafter, the present invention will be described in more detail by describing production examples and test examples as examples. Production Example 1 Natural wakame seaweed collected from January to June 1998 in Ogeshima area, Naruto City, Tokushima Prefecture was thoroughly washed to obtain a sample. 2.4 kg of leaf parts of wakame seaweed collected are finely chopped, 0.5 L Tris-HCl buffer (pH 7.9) 24 L
After stirring (37 ° C., 36 hours), the mixture was suction filtered with a glass filter (G-3). The filtered paste solution was discarded, deionized water was added to the obtained precipitate, and the mixture was homogenized and then freeze-dried to obtain a protein derived from wakame seaweed. The results of the general analysis of the protein powder derived from wakame seaweed showed that the water content was 2.8 g / 100 g and the protein was 74.1 g / 100.
g, lipid 2.1 g / 100 g, sugar 10.4 g / 100
g /, fiber 9.1 g / 100 g, ash content 1.5 g / 100
It was g. To the protein solution prepared by adding 250 mL of deionized water to 10 g of the protein powder derived from wakame prepared as described above, 300 mg each of Protease S Amano or Proleather FG-1 manufactured by Amano Pharmaceutical Co., Ltd. was added, and the pH of the protein solution containing Protease S Amano was adjusted to 8 After adjusting the pH of the protein solution containing Proleather FG-1 to 10.0, enzymatic decomposition was carried out while stirring at 65 ° C. for 5 hours.
The decomposition reaction solution was immediately subjected to an ultrafiltration membrane (YM1 manufactured by Amicon).
Type 0; the flow-through liquid passed through a molecular weight cut-off of about 10,000)
wex50W × 4 [H ⁺ ] column (φ4.0 × 55c
m). The column was thoroughly washed with deionized water and then eluted with 2 L of 2N ammonium water. After removing ammonia by concentration under reduced pressure, the concentrate was pre-buffered with deionized water to Sephadex G-25 (φ
1.6 × 113 cm), flow rate 12 mL / hr,
Gel filtration was performed with each fraction amount of 5.7 ml. After gel filtration chromatography, the fractions with high ACE inhibitory activity were collected, concentrated under reduced pressure, and then SP-Sep buffered with deionized water in advance.
hadexC-25 [H ⁺ ] column (φ1.8 × 40c
m), a concentration gradient method of 500 ml of 1.5% sodium chloride from 500 ml of deionized water, and a flow rate of 70 m
Chromatography was performed with L / hr and each fraction amount of 10 mL. After chromatography, the fractions having high ACE inhibitory activity were collected and lyophilized to obtain purified peptide powder.
After dissolving 20 mg of this purified peptide powder in 60 μL of deionized water, high performance liquid chromatography (HPL
C) was performed. The column is Nomura Chemical's Devel
osil ODS-5 (4.5EmmID x 25cm
L) is used as a mobile phase, and a concentration gradient method of 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% TFA is performed, and a flow rate is 1.0 mL / min. HPLC was performed at a detection wavelength of 220 nm to obtain a peptide fragment derived from the protein solution containing protease S Amano at an elution time of 34.5 minutes.
Peptide fragments having high ACE inhibitory activity were obtained as peptide fragments derived from the protein solution containing Proleza FG-1 at an elution time of 40.7 minutes. The amino acid sequence of the thus obtained peptide having an 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 wakame peptide having a dipeptide structure represented by L-isoleucyl-L-tyrosine. It is a white powder at room temperature.
When the dipeptide of the present invention, that is, the novel wakame peptide is used as an ACE inhibitor and is formulated into tablets, for example, it may be treated according to a conventional method, for example, as follows: wakame peptide 11 g, lactose 67 g, corn starch 38 g , Magnesium stearate 1.3g
First, and 21 g of cornstarch were mixed and granulated with a paste made from 12 g of cornstarch, 8 g of cornstarch and was added to the granules, and the resulting mixture was compressed with a compression tablet machine to give tablets. 1000 pieces are manufactured.

Production Example 2 This example is an example of production by a synthetic method. Method for synthesizing L-isoleucyl-L-tyrosine The dipeptide was synthesized by a solid phase method using an automated peptide synthesizer type 430A manufactured by Applied Biosystems. A resin obtained by chloromethylating a styrene divinylbenzene copolymer (polystyrene resin) was used as the solid phase carrier. First, according to the amino acid sequence of the dipeptide, tyrosine on the C-terminal side was reacted with chloromethyl resin according to a conventional method to obtain a peptide-bonded resin. The amino acid at this time is t-butoxycarbonyl (hereinafter, t-butoxycarbonyl).
It is abbreviated as Boc. ) Group protected t-Boc amino acid was used. Next, this peptide-bonded resin was suspended in a mixed solution of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, and trifluoroacetic acid was added under ice cooling,
Stir for a further 10 minutes. Trifluoromethanesulfonic acid was added dropwise to this mixture, and the mixture was stirred at room temperature for 30 minutes, anhydrous ether was added to precipitate and separate the product, and the precipitate was washed several times with anhydrous ether, and then under reduced pressure. Dried in. The unpurified synthetic peptide thus obtained was dissolved in distilled water or methanol and then purified by HPLC using a reverse phase column C ₁₈ (5 μm). (A) distilled water containing 0.1% TFA as mobile phase, (B)
Using a 0.1% TFA-containing acetonitrile solution,
The liquid (A) was subjected to chromatography at a flow rate of 20.1 mL / min by the concentration gradient method of 83% → 40.5% in 65 minutes. The elution fraction showing the maximum absorption, which was detected at an ultraviolet wavelength of 221 nm, was collected and freeze-dried to obtain the target synthetic dipeptide.

The synthetic dipeptide was confirmed to be a wakame peptide having a dipeptide structure represented by L-isoleucyl-L-tyrosine in amino acid composition by mass spectrum analysis, amino acid analysis and amino acid sequencer. The dipeptide according to the present invention obtained by synthesis, that is, the novel wakame peptide, was confirmed to have a pharmacological effect by the following tests.

Test Example 1 (Method for measuring angiotensin converting enzyme inhibitory activity) ACE
(Manufactured by Sigma, Enzyme No. EC3.4.5.15.1) 2.5
mU, synthetic substrate Hippuryl-L-histidy
1-L-leucine (manufactured by Peptide Institute) 12.5
The method of Yamamoto et al., which improved the measurement method of Lieberman using mM [Nippon Chinkai, Vol. 18, 297-302 (1)
989)]. That is, the produced hippuric acid was extracted with ethyl acetate and the absorbance was measured at 225 nm. The absorbance in the test solution was Es, the value when the buffer solution was added instead of the test solution was Ec, and the value when the reaction stop solution was added in advance and the reaction was Eb, and the inhibition rate was calculated from the following equation. . 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). The dipeptide of the present invention; L-isoleucyl-L-tyrosine has an inhibitory activity (IC ₅₀ value) of 2.65 μM against angiotensin converting enzyme of bovine lung serum.
Is.

Test Example 2 (Hypotensive effect when administered to spontaneously hypertensive rats) As experimental animals, 15-week-old male spontaneously hypertensive rats (hereinafter abbreviated as SHR) were purchased from Charles River Japan.
After preliminarily breeding for 1 week, 6 animals having a systolic blood pressure of 160 mmHg or more (body weight 280 to 330 g) were used as one group. Rats were housed in individual cages for rats made of stainless wire one by one in a breeding room adjusted to room temperature of 23 ± 2 ° C., humidity of 55 ± 10%, and 12 hours of light and dark (lights from 6 am to 6 pm). . MF powder feed manufactured by Oriental Yeast Co., Ltd. was used as the feed, and drinking water was self-pumped (conforming to tap water quality standards)
Each was freely ingested. Blood pressure was measured using a non-invasive blood pressure measuring device for tail artery (PS-100 type, manufactured by Riken Development Co., Ltd.).
According to the ail-cuff method, SHR was set as 0 hour before the administration, 27 hours after the administration, and 9 weeks after the administration.
Tail arterial systolic blood pressure (mmHg, upper value), mean blood pressure (m
mHg) and diastolic blood pressure (mmHg, lower value) are measured 5 times each at regular intervals, the highest and lowest values obtained are rejected, and the average value of 3 measurements is taken for each hour. And The results of the respective blood pressure values (mmHg) obtained when the synthetic dipeptide of the present invention; L-isoleucyl-L-tyrosine 10 mg / kg was orally administered to SHR for up to 27 hours are shown in FIG. Also, a synthetic dipeptide according to the present invention; L-isoleucyl-L-tyrosine 10 mg
The results for each blood pressure value (mmHg) when orally administered / kg to SHR for up to 9 weeks are as shown in FIG. As a result of these tests, it was confirmed that the dipeptide of the present invention, that is, the novel wakame peptide has angiotensin converting enzyme inhibitory activity and exhibits a significant hypotensive action in vivo (in vivo). Therefore,
The dipeptide of the present invention, that is, the novel wakame peptide is useful as a therapeutic or prophylactic agent for hypertension. The dipeptide of the present invention, that is, L-isoleucyl-L-tyrosine, can be used in the structure of a peptide structurally having the amino acid sequence as a partial structure.

[0014]

[Brief description of drawings]

1 Wakame peptide (synthetic dipeptide; L-isoleucyl-L-tyrosine) obtained in Production Example 2 10 mg /
It is a figure which shows the change of the systolic blood pressure value, the mean blood pressure value, and the diastolic blood pressure value (mmHg) when kg is orally administered to SHR for up to 27 hours.

FIG. 2 Wakame peptide (synthetic dipeptide; L-isoleucyl-L-tyrosine) obtained in Production Example 2 10 mg /
It is a figure which shows the change of the systolic blood pressure value, the mean blood pressure value, and the diastolic blood pressure value (mmHg) when kg is orally administered to SHR for up to 9 weeks.

Claims (2)

[Claims] 1. A novel wakame peptide having a dipeptide structure represented by L-isoleucyl-L-tyrosine. 2. A hypotensive agent comprising a novel wakame peptide having a dipeptide structure represented by L-isoleucyl-L-tyrosine as an active ingredient.