JPH07313185A - Production of peptide - Google Patents

Abstract

PURPOSE:To obtain a peptide effective for suppressing hypertension by inhibiting angiotensin I converting enzyme exhibiting hypertensive action in a renin- angiotensin system. CONSTITUTION:A peptide having Val-Arg-Pro structure, a peptide having Ile- Lys-Pro structure and a peptide having Leu-Arg-Pro structure are produced by subjecting fish to autodigestion and separating the product by chromatography. These peptides exhibit high inhibiting action on angiotensin I converting enzyme. A hypotensive peptide originated from natural source can easily be produced by this process.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an angiotensin I converting enzyme inhibitory peptide. For more details,
The present invention relates to an angiotensin I-converting enzyme inhibitory peptide that is an enzyme that converts angiotensin II, which functions to increase blood pressure, from angiotensin I.

[0002]

BACKGROUND ART Hypertension has a maximum blood pressure of 160 mmHg.
The minimum blood pressure is 95 mmHg or higher, or both of them are higher. In Japan, the number of patients is said to be about 20 million, which is a highly prevalent disease. Hypertension is known to cause various complications over a wide range of organs such as cerebral hemorrhage, cerebral infarction, subarachnoid hemorrhage, angina, myocardial infarction, renal sclerosis, renal failure, and retinal vein occlusion, Effective therapeutic agents are desired.

As one of the mechanisms for controlling blood pressure in the living body, there are a renin-angiotensin system which is a pressor system and a kallikrein-quinine system which is a hypotensive system. In the renin-angiotensin system, the enzyme renin is produced in renal glomerular cells (JG cells) and acts on angiotensinogen, which is a renin substrate in blood vessels, to produce angiotensin I. The enzyme that converts angiotensin I into angiotensin II is an angiotensin I converting enzyme, and the resulting angiotensin II acts on arterioles to cause contraction.

[0004] Angiotensin II also acts on the adrenal cortex, promotes the synthesis and secretion of aldosterone, promotes sodium reabsorption in the kidney, and maintains the body fluid volume. Thus, angiotensin II raises blood pressure. On the other hand, in the kallikrein-quinine system,
The proteolytic enzyme kallikrein acts on the substrate, kininogen, to produce quinine. Kinins have the functions of dilating blood vessels and lowering blood pressure, but they are degraded by kininase II. Kininase II is known to be the same substance as angiotensin I converting enzyme.

From the above, treatment of hypertension by inhibiting angiotensin I-converting enzyme is considered, and to date, captopril, enalapril, alacepril,
Delapril and others are being developed. However, although captopril has a strong antihypertensive effect, it is expensive and difficult to obtain easily. Moreover, inadequate dosage results in renal dysfunction and hypotension. Further, it is said that the SH group existing in the molecule causes rash and taste abnormality.

[0006] On the other hand, it is known that peptides derived from food proteins also have an action of inhibiting angiotensin I converting enzyme. For example, from a hydrolyzate of corn zein with thermolysin, Leu-Arg-P
Peptides with the structure of ro (Miyosi et al., Agr
ic. Biol. Chem. Volume 55, Issue 5, 131
Pp. 3 to 1318 (1991)), and a peptide having a structure of Ile-Lys-Pro has been isolated from a thermolysin degradation product of Katsuobushi (Japanese Patent Laid-Open No. 4-69396). However, since these peptides are obtained by enzymatic hydrolysis of food proteins, it is necessary to add an expensive proteolytic enzyme to the production.
Further, it has been reported that a synthetic peptide Val-Arg-Pro containing an amino acid sequence of human κ-casein also has a strong angiotensin I-converting enzyme inhibitory action (Ko.
hmura et al., Agric. Biol. Chem. Fifth
Vol. 4, No. 3, pp. 835-836 (1990)). However, since synthetic peptides are synthesized using a large number of reagents, it is necessary to remove these residual reagents before administration to the human body, which requires a great deal of labor for peptide purification.

[0007]

An object of the present invention is to provide a method for inexpensively producing an angiotensin I converting enzyme inhibitory peptide derived from a natural product from unused resources such as the internal organs of fish and shellfish.

[0008]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to solve the above problems, and as a result, completed the present invention. That is, the present invention provides a method for producing an angiotensin I converting enzyme inhibitory peptide which is inexpensive and can be easily prepared. Strong angiotensin I
Peptide having converting enzyme inhibitory action Val-Arg-P
ro, Ile-Lys-Pro and Leu-Arg-Pr
o is contained in the autolysates of the heads and internal organs of seafood and can be easily purified by the following method. The head and internal organs of seafood produced in the seafood processing industry are crushed with a crusher and self-digested at 20 to 70 ° C. Reaction time is 1
~ 6 hours is appropriate. To help digest protein
It is also possible to carry out the reaction by adding a proteolytic enzyme agent for food such as papain at this time. The proteolytic enzyme is inactivated by heating it to 90 ° C. or higher, and the solid content is removed by centrifugation or filtration. This self-digesting solution is adsorbed on a packing material having an octadecylsilyl group as a functional group (for example, Seppack C ₁₈ , manufactured by Waters), washed with water, and then eluted with an aqueous solution containing a polar solvent such as acetonitrile. The eluate is adsorbed on a cation exchanger (for example, Toyopack SP, manufactured by Tosoh Corporation), washed with a pH 6 buffer, and then eluted with a pH 8 buffer. Then, the peptide of interest can be recovered by purifying the peptide by reverse phase chromatography and / or gel filtration chromatography. As the fish species that can be used in the present invention, skipjack, which produces a large amount of offal in the bonito industry, is easy to use, but other fish species such as sardines and tuna can also be used.

[0009]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 1 kg of skipjack internal organs was crushed with a crusher to give 500 g.
Water was added, and the autolysis reaction was carried out at 50 ° C. for 3 hours while gently stirring. Then, it boiled for 20 minutes, the solid content was removed by centrifugation, and ultrafiltration was performed using a laboratory module ultrafiltration system SIP-1013 manufactured by Asahi Kasei. Reverse-phase pretreatment cartridge, Sepppack C ₁₈ ENV (manufactured by Waters)
Adsorbed on and washed with 8 ml of distilled water, then acetonitrile 1
Elution was carried out with 8 ml of a 5% aqueous solution. This process was repeated 4 times, and the acetonitrile eluate was mixed. The solvent was removed from this solution under reduced pressure, and a 10 mM phosphate buffer solution (pH 6.
After adsorbing on the ion exchange pretreatment cartridge, Toyopack IC-SP (M) (manufactured by Tosoh Corporation) equilibrated with 0), the sample was washed with 4 ml of the same buffer and eluted with 4 ml of 10 mM dipotassium hydrogen phosphate solution. . Then, separation was performed by reversed-phase high performance liquid chromatography under the conditions shown in HPLC condition 1. The result at this time is shown in FIG. Fraction 1 of Chart 1 was further subjected to the HPLC conditions 2
Separation was performed by reversed-phase high performance liquid chromatography. The result is shown in FIG. Fraction 1-1 of Figure 2 was further subjected to HPLC
Separation was performed by gel filtration chromatography under condition 3. The result is shown in FIG. Fractions 1-2 of Figure 3 are further HPL
It was separated by gel filtration chromatography under C condition 4. The result is shown in FIG. A strong angiotensin I-converting enzyme inhibitory action was observed in Fractions 1-3 in FIG. 4, so a gas phase protein sequencer 4 manufactured by Applied Biosystems
When structural analysis was performed with 70A, Var-Arg-P
It was found to be the structure of ro.

The measurement of the inhibition of angiotensin I converting enzyme is carried out by the method of Maruyama et al. (Agricultural Biological Chemistry, vol. 46, 5) which is an improved version of the method of Kashman et al. No. 1393-1394 (1
982)). That is, to a test tube, 30 μl of an aqueous solution of the peptide of the present invention and 250 μl of a borate buffer solution of pH 8.3 containing L-hipryl histidyl leucine (manufactured by Sigma) and sodium chloride as an enzyme substrate were added,
Pre-incubated at 10 ° C for 10 minutes. Then, 100 μl of angiotensin I converting enzyme-containing solution was added to start the enzymatic reaction. At this time, the borate buffer solution had a concentration of 0.
1M, L-Hipryl histidyl leucine concentration is 5 mM,
Sodium chloride is 300 mM, and the enzyme activity without inhibition is 8 mU. 3 at 37 ° C, pH 8.3
After reacting with shaking for 0 minutes, 250 μl of 1N hydrochloric acid
Was added to stop the reaction. 1.5 ml of ethyl acetate was added, and the mixture was shaken for 15 seconds to extract hippuric acid generated by the enzymatic reaction, and centrifuged at 2000 rpm for 10 minutes to recover an ethyl acetate layer 1.0 in a test tube. Ethyl acetate was heated in a hot dry bath at 120 ° C. for 30 minutes to completely remove it, and then left at room temperature for 5 minutes. Then, 3 ml of distilled water was added, and the amount of hippuric acid produced was determined by measuring the 228 nm absorbance. The angiotensin I-converting enzyme-containing solution used for the enzymatic reaction was 1 g of rabbit rung acetone powder (manufactured by Sigma) and 0.1 M borate buffer solution (pH 8.3).
Dissolve in 10 ml and stir well, then 4 ℃, 40000
It was prepared by diluting the supernatant after centrifugation at xg for 40 minutes with 0.1 M borate buffer. Further, hydrochloric acid was added before the start of the enzymatic reaction, and the same operation was performed, but the measurement result was used as a blank. When 2.2 μM of the peptide was added to the reaction solution, the activity of angiotensin I converting enzyme was inhibited by 50%.

Example 2 Fraction 2 obtained by reverse phase chromatography of Example 1
(FIG. 1) was further separated under the condition of HPLC condition 2. The results are shown in Fig. 5. The fraction 2-1 in FIG.
Separated by gel filtration chromatography under LC condition 3.
The result is shown in FIG. Fraction 2-2 in FIG.
Separation was performed by gel filtration chromatography under PLC condition 4. The result is shown in FIG. 7. Fraction 2-3 in FIG. 7 was further subjected to ion exchange separation under HPLC condition 5 to obtain fraction 2-4 in FIG. This fraction was finally subjected to reverse phase system separation under HPLC condition 6 to obtain fraction 2-5 shown in FIG. Fractions 2-5 were found to have a strong angiotensin I converting enzyme inhibitory action. Therefore, after desalting, structural analysis was performed using a gas phase protein sequencer 470A manufactured by Applied Biosystems, and it was found that the structure was Ile-Lys-Pro. It turned out to be. When the angiotensin I-converting enzyme inhibitory activity of the present peptide was measured by the method described in Example 1,
When this peptide was added to the reaction solution at 2.5 μM, the activity of angiotensin I converting enzyme was inhibited by 50%.

Example 3 Fraction 3 obtained by reverse phase chromatography of Example 1
(FIG. 1) was further separated under the condition of HPLC condition 2. The results are shown in Fig. 10. Fraction 3-1 in FIG. 10 was further separated by gel filtration chromatography under HPLC condition 3. The result is shown in FIG. Fraction 3-2 in FIG. 11 was further separated by gel filtration chromatography under HPLC condition 4. The result is shown in FIG. Fraction 3 in Figure 12
-3 is further subjected to ion exchange separation under HPLC condition 5,
Fractions 3-4 shown in FIG. 13 were obtained. HP at the end of this fraction
Reversed-phase separation was performed under LC condition 6, and fractions 3-shown in FIG.
Got 5. Since a strong angiotensin I-converting enzyme inhibitory action was observed in Fractions 3-5, after desalting, structural analysis was performed using a gas phase protein sequencer 470A manufactured by Applied Biosystems, and it was found that the structure was Leu-Arg-Pro. It turned out to be. When the angiotensin I-converting enzyme inhibitory activity of the peptide was measured by the method described in Example 1, the activity of the angiotensin I-converting enzyme was 50 when the peptide was added to the reaction solution at 1.0 μM.
% Inhibited.

HPLC condition 1 Column: RP-18 (e) 10 mm ID × 250 mm
L (manufactured by Merck) Mobile phase: A 0.05% trifluoroacetic acid solution B: 0.05% trifluoroacetic acid, 30% acetonitrile solution Gradient was 100% of solution A and 120 minutes later, solution B 10
0% linear gradient Flow rate: 4.0 ml / min Detection: UV210 nm

HPLC condition 2 Column: RP-18 (e) 4.6 mm ID × 250 m
mL (manufactured by Merck) Mobile phase: A 0.05% trifluoroacetic acid solution B: 0.05% trifluoroacetic acid, 30% acetonitrile solution Gradient was 100% of solution A and 120 minutes later, solution B 10
0% linear gradient Flow rate: 1.0 ml / min Detection: UV210 nm

HPLC condition 3 column: GS-220 7.6 mm ID × 500 mm L
(Asahi Kasei Co., Ltd.) Mobile phase: 50 mM ammonium acetate Flow rate: 1.0 ml / min Detection: UV 210 nm

HPLC condition 4 Column: GS-320 7.6 mm ID × 500 mm L
(Asahi Kasei Co., Ltd.) Mobile phase: 50 mM ammonium acetate Flow rate: 1.0 ml / min Detection: UV 210 nm

HPLC condition 5 Column: TSKgel SP-2SW 4.6 mm ID
× 250 mmL (manufactured by Tosoh Corporation) Mobile phase: A 20 mM sodium phosphate buffer (pH
6.0) B 20 mM sodium phosphate buffer (pH 6.0) +
0.5M sodium chloride A 100% A flow for 10 minutes, and then 40 minutes later, a linear gradient such that the solution B becomes 100% Flow rate: 0.8 ml / min Detection: UV210 nm

HPLC condition 6 Column: RP-18 (e) 4.6 mm ID × 250 m
mL (manufactured by Merck) Mobile phase: 0.05% trifluoroacetic acid, 7% acetonitrile solution Flow rate: 1.0 ml / min Detection: UV210 nm

[0019]

As described above, according to the present invention,
An angiotensin I converting enzyme inhibitory peptide having an action of suppressing an increase in blood pressure can be easily obtained without adding a proteolytic enzyme agent.

[Sequence list]

SEQ ID NO: 1 Sequence length: 3 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide

SEQ ID NO: 2 Sequence length: 3 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide

SEQ ID NO: 3 Sequence length: 3 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide

[Brief description of drawings]

FIG. 1 shows a peptide solution derived from skipjack self-digested product as RP-1.
8 (e) Elution pattern when separated with a column (10 mm ID × 250 mm L).

FIG. 2 is an elution pattern when fraction 1 in FIG. 1 is separated under HPLC condition 2.

FIG. 3 is an elution pattern when fraction 1-1 of FIG. 2 is separated under HPLC condition 3.

FIG. 4 is an elution pattern when fractions 1-2 of FIG. 3 are separated under HPLC condition 4.

FIG. 5 is an elution pattern when Fraction 2 of FIG. 1 is separated under HPLC condition 2.

FIG. 6 is an elution pattern when the fraction 2-1 of FIG. 5 is separated under HPLC condition 3.

FIG. 7 is an elution pattern when fraction 2-2 in FIG. 6 is separated under HPLC condition 4.

8 is an elution pattern when fractions 2-3 in FIG. 7 were separated under HPLC condition 5. FIG.

9 is an elution pattern when fractions 2-4 in FIG. 8 are separated under HPLC condition 6. FIG.

FIG. 10 is an elution pattern obtained when Fraction 3 in FIG. 1 was separated under HPLC condition 2.

FIG. 11 is an elution pattern when the fraction 3-1 of FIG. 10 is separated under HPLC condition 3.

FIG. 12 is an elution pattern obtained by separating the fraction 3-2 shown in FIG. 11 under HPLC condition 4.

FIG. 13 is an elution pattern when fractions 3-3 in FIG. 12 are separated under HPLC condition 5.

FIG. 14 is an elution pattern when fractions 3-4 in FIG. 13 are separated under HPLC condition 6.

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display area A61K 38/55 AEQ C07K 123: 00

Claims (3)

[Claims] 1. A method for producing an angiotensin I converting enzyme inhibitory peptide, which comprises autolyzing seafood and separating and recovering a peptide having a Val-Arg-Pro structure from the autolysate. 2. A method for producing an angiotensin I converting enzyme inhibitory peptide, which comprises autolyzing seafood and separating and recovering a peptide having a structure of Ile-Lys-Pro from the autolyzed product. 3. A seafood is self-digested, and L is extracted from the self-digested product.
A method for producing an angiotensin I converting enzyme inhibitory peptide, which comprises separating and collecting a peptide having a structure of eu-Arg-Pro.