JP2920829B1 - Novel pentapeptide and angiotensin converting enzyme inhibitors - Google Patents

Abstract

Abstract: [Object] To provide a novel pentapeptide having angiotensin converting enzyme inhibitory activity from a digestion solution of ginseng protease. The pentapeptide Ile-Gly-Pro-Ala-Gly has a novel angiotensin-converting enzyme inhibitory effect by treating ginseng with a protease or the like.
It has a blood pressure lowering effect in vivo and has extremely low toxicity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pentapeptide having a peptide structure of the following amino acid sequence, which is useful as a pharmaceutical, and an angiotensin converting enzyme inhibitor containing the pentapeptide as an active ingredient. Ile-Gly-Pro-Ala-Gly (wherein each symbol representing an amino acid residue is represented by a convention used in amino acid chemistry.)

[0002]

BACKGROUND OF THE INVENTION It is well known that the renin-angiotensin system plays an important role in the regulation of water, electrolytes and blood in living organisms. An angiotensin converting enzyme (hereinafter abbreviated as ACE) exists in the renin-angiotensin system, and angiotensin I is converted into angiotensin I by ACE.
Converted to I. Angiotensin II is a powerful vasopressor and acts on the central nervous system and peripheral nervous system as well as blood vessels and the adrenal cortex to promote blood pressure elevation. ACE has a function of decomposing and inactivating bradykinin, which is a hypotensive substance in a living body, and is involved in the pressor system. Therefore, it is possible to lower blood pressure by inhibiting the activity of ACE, and it is considered that this is clinically effective for prevention and treatment of hypertension. For this purpose, a proline derivative, captolyl, was synthesized and its antihypertensive action was confirmed.Since then, various ACE inhibitors have been actively studied for synthesis based on the structural study of captolyl, and recently enalabryl maleate and alacebryl have been developed. Are being provided to clinical sites one after another. At present, ACE inhibitors are used for hypertension, regardless of essential hypertension, symptomatic hypertension,
In addition, it is widely used in both mild and severe cases and is added to the first-line treatment of hypertension, and has been found to have many excellent points. On the other hand, as the mechanism of action of the ACE inhibitor, suppression of aldosterone and vasopressin secretion by suppressing the production of angiotensin II and promotion of sodium and water excretion by releasing renal artery contraction are considered. Furthermore, it is believed that the ACE inhibitor suppresses the inactivation of the kallikren-kinin system and activates the prostaglandin system to further promote peripheral vasodilation and excretion of sodium and water,
It is expected as a suitable therapeutic drug in breaking the vicious cycle of heart failure. By the way, as an ACE inhibitor, in addition to the above-mentioned synthetic products, a natural product or a substance derived from a natural product, a snake venom-derived bradykinin enhancing factor (C-terminal is Pro) [S.
H. Ferreia et al: Biochemis
try, 9, 3583 (1970)], and six types of peptides derived from collagenase digested gelatin (Ala-Hyp at the C-terminus) [G. Oshima et a
1: Biochim. Biophs. Acta, 56
6, 128 (1979)], a peptide derived from a tryptic digest of bovine casein (Gly-Lys at the C-terminus) [S.
See Maruyama et al. : Agric. Bio
l. Chem. , 46, 1393 (1983)], and five other hexapeutides derived from the sardine muscle of the present inventors (in each case, the second or third from the C-terminal is Pro, N
The terminal is Leu) [Patent No. 2046483], a laver-derived tetrapeptide (N-terminal is Pro) [Patent No. 2678]
No. 180], and it is disclosed that any of them can be an ACE inhibitor. Furthermore, di- and tripeptides having a short chain length obtained by a synthesis method [Japanese Patent Application Laid-Open No. 6-87886].
Although Japanese Patent Application Laid-Open No. 6-16568 has been proposed, the ACE inhibitory effect of a peptide having a long chain length having a regular amino acid sequence and the antihypertensive effect (pharmacological effect) by oral administration are unknown. Although it has been a long time since its discovery, there are no reports that it is still being developed as a drug. In the case of food, Suzuki et al.
ACE inhibitory activity has been observed in shellfish and fruits [Takeo Suzuki, Nobuko Ishikawa et al .;
3)] However, it has not been known that ginseng as a medicinal plant (Chinese medicine) has an ACE inhibitor.

[0003]

Means for Solving the Problems The present inventor has conducted intensive studies to solve the above-mentioned problems, and as a result, a medicinal plant (Chinese medicine)
The present inventors have found that the novel pentapeptide of the present invention obtained from Korean ginseng as described above has a blood pressure lowering effect, and have completed the present invention. That is, a substance having a pharmacological action was searched for from the digested solution of ginseng proteolytic enzyme, and it was found that the novel pentapeptide had a strong angiotensin converting enzyme inhibitory action. Intensive research has been conducted on the practical use of this pentapeptide as a drug, and as a result, it has been found that this pentapeptide has a blood pressure lowering effect and is useful as an angiotensin converting enzyme inhibitor derived from natural products. The present invention is based on such findings. The novel peptide according to the present invention is a novel pentapeptide represented by an L-form amino acid sequence represented by the following formula Ile-Gly-Pro-Ala-Gly, and is a white powder at room temperature.

The novel pentapeptide can be chemically synthesized or separated and purified from a digestion solution of ginseng protease. When the novel pentapeptide according to the present invention is chemically synthesized, it can be performed by a conventional synthesis method such as a liquid phase method or a solid phase method. L corresponding to the amino acid residue from the C-terminal side (carboxyl-terminal side) of the pentapeptide to the support
The amino acids of the body are preferably linked sequentially by peptide bonds. Then, the synthetic pentapeptide thus obtained is cleaved from the poromerous solid support using trifluoromethanesulfonic acid, hydrogen fluoride, or the like, and then the protecting group of the amino acid side chain is removed. It can be purified by an ordinary method using high performance liquid chromatography (hereinafter abbreviated as HPLC) using a system column.

[0005] As described above, the novel pentapeptide according to the present invention can be separated and purified from the digestion solution of ginseng proteolytic enzymes. In such a case, it can be carried out as follows. it can. Hydrolysis is performed using ginseng containing the above novel pentapeptide. The hydrolysis is performed according to a conventional method. For example, when hydrolyzing with a proteolytic enzyme such as pepsin, the ginseng homogenate is further hydrolyzed if necessary, and then heated to the optimal temperature of the enzyme to adjust the pH to the optimal value, and the enzyme is added. Incubate. Then, if necessary, after neutralization,
Deactivate the enzyme 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 semi-permeable membrane such as cellophane to a suitable solvent (eg, water, Tris-hydrochloric acid). Buffer, phosphate buffer, neutral buffer, etc.), and the solution containing the components in the filtrate that has passed through the semipermeable membrane is converted to a strongly acidic cation exchange resin (for example, Dow Chemical Co., Ltd.). The product is subjected to an angiotensin converting enzyme (hereinafter referred to as A).
A fraction containing a component having an inhibitory activity (abbreviated as CE) is obtained, and the obtained ACE inhibitory activity fraction is subjected to gel filtration (for example, Sephadex G-25 manufactured by Pharmacia).
And the obtained ACE inhibitory activity fraction is fractionated by cation exchange gel filtration (for example, SP-Scphadex C-25 manufactured by Pharmacia), and the obtained ACE inhibitory activity fraction is obtained. Is further reversed phase HPL
C can be performed by fractionation.

[0006] As the Ukogiaceae plant used in the method for producing the novel pentapeptide according to the present invention, any Ukogiaceae plant may be used as long as the object of the present invention can be achieved, but preferably Ginseng (also known as Panax ginseng and Korean ginseng) Carrot) is preferably used. The novel pentapeptide of the present invention obtained as described above does not induce antibody production and does not cause anaphylactic shock when repeatedly administered intravenously. In addition, the novel pentapeptide of the present invention has a sequence structure of only L-amino acids and is gradually degraded by an in-vivo protease after administration. Therefore, the toxicity is extremely low and the safety is extremely high (LD
₅₀ > 5000 mg / kg; oral administration to rats). The novel pentapeptide 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 usually includes injection into mammals (eg, humans, dogs, rats, etc.) having ACE, or oral administration. The dose is, for example, 0.01 to 10 mg of the pentapeptide per animal body weight. The number of administrations 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 above-mentioned various preparations are not particularly limited, and those used in usual injections, powders, granules, tablets or capsules are used. Can be. Examples of additives used for tablets, capsules, granules, and powders include the following. As excipients, sugars such as crystalline cellulose, sugar alcohols such as mannitol, starch, anhydrous calcium phosphate, etc .; starches, hydroxypropylmethyl cellulose, etc. as binders; carboxymethylcellulose and its potassium salts as disintegrants; Stearic acid and its salts as lubricants,
Examples include talc and waxes. In preparing the preparation, a odorant such as menthol, citric acid and its salts, and a fragrance can be used as necessary. The sterile composition for injection is prepared by dissolving or suspending the novel pentapeptide 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 an ordinary method. It can be turbid to obtain an injection. At this time, buffers, preservatives, antioxidants and the like can be added as necessary. The preparation containing the novel pentapeptide of the present invention may be in the form of a lyophilized product or a dry powder, and may be used by dissolving it in a usual dissolving agent, for example, water or physiological saline when used.

[0008] The novel pentapeptide according to the present invention has an excellent angiotensin converting enzyme inhibitory action, and exhibits a blood pressure lowering action and an inhibitory action on bradykinin inactivation. Therefore, a preventive or therapeutic agent for hypertension such as essential hypertension, renal hypertension, adrenal hypertension, and the like, which has an effect of normalizing organ circulation and improving long-term prognosis (life-extending effect) for congestive heart failure and treating heart failure Useful as an agent.

[0009]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which are Production Examples and Test Examples. Production Example 1 Deionized water (1 l) was added to 200 g of ginseng, homogenized, and the pH was adjusted to 2.0 with 1N hydrochloric acid.
g was added and hydrolysis was carried out while stirring at 37 ° C. for 20 hours. The decomposition reaction solution was immediately passed through an ultrafiltration membrane (Amicon,
(YM10, φ76mm) and the passing liquid is Dow
ex50WX4 [H ⁺ ] column (φ4.5 × 15cm)
Added. After thoroughly washing the column with deionized water,
Elution was performed using 2N ammonium hydroxide solution 2ι. Ammonia was removed by concentration under reduced pressure to obtain a concentrated solution (40 ml). 4 ml of this concentrated solution was previously buffered with deionized water.
The solution was loaded on ephadex G-25 (φ2.5 × 150 cm), and gel filtration was performed at a flow rate of 30 ml / hr and a fractionation amount of 8.6 ml. The result is as shown in FIG. The gel filtration was repeated, and fractions having high ACE inhibitory activity collected in large amounts were collected and freeze-dried to obtain peptide powder. After dissolving 3 g of this peptide powder in 20 ml of deionized water, SP-Sephadex C-25 previously buffered with deionized water.
[H ⁺ ] column (φ1.5 × 47.2 cm) was loaded,
A concentration gradient method was carried out from 1 l of deionized water to 1 l of 3% sodium chloride, and chromatography was carried out at a flow rate of 30 ml / hr and a fractionation volume of 10 ml. The result is as shown in FIG. In the above chromatograph, A of fraction numbers 25 to 37
The CE inhibitory activity fractions were collected and freeze-dried to obtain a purified peptide powder (SP-2 fraction). This purified peptide powder 20
mg was dissolved in 60 μl of deionized water, followed by high performance liquid chromatography (HPLC). As the column, Develosil ODS-5 (4.
5 mm IDX 25 cmL), using a concentration gradient method of 0.05% trifluoroacetic acid (hereinafter abbreviated as TFA) to 25% acetonitrile / 0.05% TFA as a mobile phase, and a flow rate of 1.0 ml / min. 2
Chromatography was performed at 20 nm to obtain a pentapeptide having an ACE inhibitory action. The result is as shown in FIG. 3, and the elution time by HPLC is 18.625 minutes. The thus obtained amino acid sequence of the pentapeptide having an ACE inhibitory action is obtained by using a protein sequencer-4 manufactured by Applied Biosystems (ABI).
Determined using 77A type. As a result, it was confirmed that the novel peptide according to the present invention was a novel pentapeptide represented by an L-form amino acid sequence represented by the following formula Ile-Gly-Pro-Ala-Gly. The property at room temperature is a white powder. When the novel pentapeptide according to the present invention is formulated as an ACE inhibitor in, for example, a tablet, it may be treated according to a conventional method, for example, as follows: peptide 13 g, lactose 87 g, corn starch 29 g, stearic acid Using 1 g of magnesium as a raw material, first, and 17 g of corn starch were mixed,
Granulate with 7 g of corn starch paste and add 5 g of corn starch to the granules,
The resulting mixture is compressed with a compression tablet machine to produce 1000 tablets.

Production Example 2 This is an example of production by a synthesis method. Synthesis Method of Ile-Gly-Pro-Ala-Gly The peptide was synthesized by a solid phase method using an automatic peptide synthesizer model 430A manufactured by Applied Biosystems. A resin obtained by chloromethylating a styrene-divinylbenzene copolymer (polystyrene resin) was used as the solid support. First, according to the amino acid sequence of the pentapeptide, a glycine on the C-terminal side was reacted with a chloromethyl resin according to a conventional method to obtain a peptide-bound resin. The amino acid at this time is t-butoxycarbonyl (hereinafter, t-butoxycarbonyl).
A t-Boc amino acid protected with a group (abbreviated -Boc) was used. Next, this peptide-bonded resin was suspended in a mixture of ethanedithiol and thioanisole, stirred at room temperature for 10 minutes, and then added with trifluoroacetic acid under ice-cooling.
Stir for another 10 minutes. Trifluoromethanesulfonic acid was added dropwise to the mixture, and the mixture was stirred at room temperature for 30 minutes. The product was precipitated by adding anhydrous ether and separated.The precipitate was washed several times with anhydrous ether, and then dried under reduced pressure. And dried. The unpurified synthetic peptide thus obtained was dissolved in distilled water, and then the reverse-phase column C ₁₈ (5 μm) was used.
Purified by HPLC using m). (A) distilled water containing 0.1% TFA as a mobile phase, (B) 0.1% TF
Using an A-containing acetonitrile solution, the solution (A) was produced at a flow rate of 1.4 ml by a concentration gradient method from 92% to 81% in 20 minutes.
/ Min chromatography. UV wavelength 2
The eluted fraction detected at 14 nm and showing the maximum absorption was collected and lyophilized to obtain the desired synthetic peptide.

Analysis of this synthetic peptide by mass spectrum confirmed that it was a pentapeptide having the amino acid sequence structure of the following formula: Ile-Gly-Pro-Ala-Gly. The result of this mass spectrum is as shown in FIG. The pharmacological effect of the pentapeptide of the present invention obtained by synthesis was confirmed by the tests described below.

Test Example 1 (Method for measuring angiotensin converting enzyme inhibitory activity) ACE
(Manufactured by Sigma, Enzyme number EC 3.4.15.1) 2.5
mU, synthetic substrate Hippuryl-L-histidy
1-L-leucine (manufactured by Peptide Research Institute) 12.5
Yamamoto et al., which improved the method of measuring Lieberman using mM [Jisho Kyoukai, 18, 297-302 (198)
9)]. That is, the generated hippuric acid was extracted with ethyl acetate and measured at an absorbance of 225 nm.
The inhibition rate was determined from the following equation, where Es was the absorbance of the test solution, Ec was the value obtained when a buffer was added instead of the test solution, and Eb was the value obtained when a reaction stop solution was added in advance to react. . Inhibition rate (%) = (Ec-Es) / (Ec-Eb) × 100 The inhibitory activity IC ₅₀ value of the ACE inhibitor is the concentration of the sample required to inhibit the enzyme activity of ACE by 50% (inhibition rate) ( M). The inhibitory activity (IC ₅₀ value) of bovine lung serum against the angiotensin converting enzyme of the novel pentapeptide according to the present invention is 27.6 μM.

Test Example 2 (Hypertensive effect upon administration to spontaneously hypertensive rats) Experimental animals were purchased from Charles River Japan, a 15-week-old male spontaneously hypertensive rat (hereinafter abbreviated as SHR).
After pre-breeding for one week, the animals were used as a group of six animals having a systolic blood pressure of 160 mmHg or more (body weight: 280 to 330 g). The rats were individually housed in stainless steel rat individual gauges in a breeding room adjusted to room temperature of 23 ± 2 ° C., humidity of 55 ± 10%, and 12 hours light / dark (light on from 6:00 am to 6:00 pm). . Feed is MF powder feed manufactured by Oriental Yeast Co., Ltd. Drinking water is self-pumped (conforming to tap water quality standards)
Was freely taken. The blood pressure was measured using a non-invasive tail arterial blood pressure measurement device (manufactured by Riken, Model PS-100).
By the aile-cuff method, before administration, 1 hour after administration, 2 hours
Measurement of the systolic blood pressure value (mmHg) of the tail artery of the SHR for 5 hours, 3 hours, 4 hours and 6 hours was performed 5 times at each measurement time, and the highest and lowest values of the obtained measurement values were discarded. The average value of the times was used as the measured value at each time. The results on the effect of orally administered 200 mg / kg of synthetic pentapeptide to SHR on systolic blood pressure (mmHg) are shown in FIG.
As shown in FIG. As a result of the above test, it was confirmed that the pentapeptide according to the present invention has an angiotensin converting enzyme inhibitory activity and exhibits a significant blood pressure lowering effect even in vivo (in vivo). Therefore, the pentapeptide according to the present invention is useful as an agent for treating or preventing hypertension. In addition, the pentapeptide according to the present invention can also be employed in the structure of a peptide having the amino acid sequence as a partial structure.

[0014]

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of separation and purification of an ACE inhibitory peptide by Sephadex G-25 column chromatography in Production Example 1 of a ginseng pepsin degradation solution according to the present invention. In the figure, 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 were used as markers.

FIG. 2 shows SP-Sephadex C-25 in Production Example 1 of a ginseng pepsin hydrolyzate according to the present invention.
It is a figure which shows the result of separation and purification of an ACE inhibitory peptide by (H ^<+> ) column chromatography.

FIG. 3 shows the results of separation and purification of an ACE inhibitory peptide by reverse phase HPLC in Production Example 1 of the pentapeptide according to the present invention.

FIG. 4 is a view showing a mass spectrum of the synthetic tetrapeptide obtained in Production Example 2 of the pentapeptide according to the present invention.

FIG. 5: Synthetic tetrapeptide 200 obtained in Production Example 2
It is a figure which shows the time-dependent change of the systolic blood pressure value (mmHg) when mg / kg is orally administered to SHR. In the figure, captopril (10 mg / kg SH) was used as a control antihypertensive.
R) and 0.9% saline 3 as a control.
ml was used.

Claims (2)

(57) [Claims] 1. The following formula: Ile-Gly-Pro-A
A novel pentapeptide having a peptide structure based on the L-amino acid sequence represented by la-Gly. 2. The following formula: Ile-Gly-Pro-A
An angiotensin converting enzyme inhibitor comprising, as an active ingredient, a novel pentapeptide having a peptide structure represented by an L-amino acid sequence represented by la-Gly.