JPH05306296A - Fish-derived peptide and its production - Google Patents

Abstract

PURPOSE:To provide a peptide capable of inhibiting angiotensin I converting enzyme having hypertensive activity with renin-angiotensin system, to be used for suppressing hypertension. CONSTITUTION:The objective peptide having Gly-His-Phe or Tyr-Arg-Pro-Tyr structure has been synthesized using solid phase or liquid phase method, a chemical synthesis method for peptides. This peptide, at concentrations of 1100muM and 380muM for the former and latter structures, respectively, can inhibit, at a level of 50%, angiotensin I converting enzyme. Thus, the objective peptide having hypotensive activity can easily be provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an angiotensin I converting enzyme inhibitory peptide and a method for producing the same. More specifically, angiotensin II, which works to raise blood pressure
The present invention relates to an inhibitory peptide of angiotensin I-converting enzyme which is an enzyme that converts angiotensin I, and a method for producing the same.

[0002]

2. Description of the Related Art Hypertension is a condition in which the maximum blood pressure is 160 mmHg or higher, the minimum blood pressure is 95 mmHg or higher, or both. In Japan, it is said that the number of patients is about 20 million, which is a highly prevalent disease. Hypertension is known to cause various complications across a wide range of organs such as cerebral hemorrhage, cerebral infarction, subarachnoid hemorrhage, angina, myocardial infarction, nephrosclerosis, renal failure, and retinal sillosis. Therefore, effective therapeutic agents are desired.

As one of the mechanisms for controlling blood pressure in the living body, there are a repressor system such as renin and angiotensin system and a hypotensive system such as kallikrein and quinine 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 synthesis and secretion of aldosterone, promotes reabsorption of sodium in the kidney, and retains 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. Kinin has the function of dilating blood vessels and lowering blood pressure.
It undergoes decomposition by ZE II. It is known that kininase II is 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. It is also known that peptides derived from casein, zein, gelatin, fish meat, etc. have a function of inhibiting angiotensin I converting enzyme. (JP-A-59-44324 and JP-A-64)
-5497, JP-A 01-313498).

[0006] 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 dysgeusia. Peptides derived from casein, zein, fish meat, etc. are derived from natural products, and produce many unused substances in the manufacturing process, which not only costs a huge amount of processing but also causes environmental pollution due to disposal. I have a concern. For example, when only fish meat is used as a raw material, the internal organs remain as an unused material. On the other hand, in the seafood processing industry, the meat portion of seafood is mainly processed, and the large amounts of internal organs are often discarded as unused materials,
It may cause environmental pollution, and new effective utilization methods are required. Also, other angiotensin I converting enzyme inhibitory peptides are required.

[0007]

Therefore, there is a need for an angiotensin I-converting enzyme inhibitor that can be obtained at low cost and has few side effects. In addition, a method for effectively utilizing the unused internal organs of seafood is also desired.

[0008]

Means for Solving the Problems The present inventors have completed the present invention as a result of various studies for solving the above problems. That is, the present invention relates to an angiotensin I converting enzyme inhibitory peptide which is inexpensive and can be easily synthesized, and a method for producing the same.

The angiotensin I-converting enzyme inhibitory peptides Gly-His-Phe and Tyr-Arg referred to in the present invention
-Pro-Tyr self-digests the head and internal organs of seafood,
It can be separated and purified, or obtained by a chemical synthesis method. That is, the internal organs of seafood produced in the seafood processing industry are crushed with a crusher and self-digested at 20 to 70 ° C. A reaction time of 1 to 6 hours is suitable. Then, the mixture is heated at 90 ° C. or higher to inactivate the enzyme, and the solid matter is removed by centrifugation or filtration. Then, after adsorbing to an ODS packing (a silica gel in which octadecyl group is chemically bonded) which is a packing material for reversed-phase partition / adsorption chromatography, washing with water, and elution with a 15% acetonitrile solution, a cation exchanger SP It is adsorbed on (a carrier having a sulfopropyl group bound thereto), washed with a buffer having a pH of 6.0, and then eluted with a solution having a pH of 9.0. Then, it is purified by reverse phase high performance liquid chromatography and gel filtration chromatography. As the fish species that can be used in the present invention, skipjack, which produces a large amount of internal organs in the bonito flakes industry, is easy to use, but other fish species such as sardines and tuna can also be used.

Further, the present peptide can be produced by various chemical synthesis methods such as solid phase method and liquid phase method.
That is, Boc (tert-butyroxycarbonyl) -amino acid is reacted with DCC (dicyclohexylcarbodiimide) and HoBt (1-hydroxybenzotriazole) in DMF (dimethylformamide), or the amino acid is condensed on an insoluble carrier by the Merrified method. The method can be used. In using the present peptide, either oral administration or intravenous injection can be used, and the peptide can also be used in the form of various salts such as hydrochloric acid and trifluoroacetic acid.

[0011]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 1 kg of bonito offal 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 substance was removed by centrifugation, and ultrafiltration was performed using the laboratory module ultrafiltration system SIP-1013 of Asahi Kasei. The peptide of the filtrate was adsorbed on a reverse phase pretreatment cartridge Seppak C ₁₈ ENV (manufactured by Waters), washed with 8 ml of distilled water, and then acetonitrile 15%
The solution was eluted with 8 ml. Repeat this process 4 times 136
mg of peptide was processed. What was eluted with 15% acetonitrile solution was 10 mM after removing the solvent under reduced pressure.
Ion exchange system pretreatment cartridge equilibrated with phosphate buffer (pH 6.0), Toyopack IC-SP (M)
(Tosoh Co., Ltd.), then washed with 4 ml of 10 mM phosphorus buffer (pH 6.0), eluted with 4 ml of 10 mM potassium dihydrogen phosphate, and subjected to reversed-phase high performance liquid chromatography under the conditions shown in HPLC condition 1. Separated by. The result at this time is shown in FIG. Peak 1 was further separated by reverse phase high performance liquid chromatography under the condition of HPLC condition 2. The result is shown in FIG. Peak 2 was further separated by gel filtration high performance liquid chromatography under HPLC condition 3. The chart at this time is shown in FIG. Of these, strong angiotensin I-converting enzyme inhibitory activity was observed at peaks 3 and 4 in FIG. 3, so structural analysis was performed using a gas phase protein sequencer 470A of Applied Biosystems.
Peak 4 was found to be the structure of Gly-His-Phe.

The measurement of angiotensin I converting enzyme inhibition is carried out by the method of Maruyama et al., Which is an improved method of Kashman et al. (Biochemical Pharmacologi Vol. 20, pp. 1637-1648 (1971)). Chemistry 46, No. 5, pp. 1393-1394 (1982)).

That is, a test tube containing 30 μl of an aqueous solution of the peptide of the present invention, L-hypril histidyl leucine (manufactured by Sigma) as an enzyme substrate, and sodium chloride was added at pH 8.
Add 250 μl of borate buffer 3 of 3 and add 10
Pre-incubated for minutes. Then, 100 μl of a solution containing angiotensin I converting enzyme was added to start the enzymatic reaction. At this time, the concentration of borate buffer is 0.1M,
The L-hypril histidyl leucine concentration is 5 mM and sodium chloride is 300 mM, and the enzyme activity in the absence of inhibition is 8 mU. After reacting with shaking at 37 ° C. and pH 8.3 for 30 minutes, 250 μl of 1N hydrochloric acid was added to stop the reaction. Add 1.5 ml of ethyl acetate,
Hippuric acid generated by the enzymatic reaction was extracted by shaking for 15 seconds, and centrifuged at 2000 rpm for 10 minutes to collect 1.0 ml of an ethyl acetate layer 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, distilled water was added, and the amount of hippuric acid produced was determined by measuring the absorbance at 228 nm. The angiotensin I-converting enzyme-containing solution used in the enzymatic reaction was a rabbit acetone powder (manufactured by Sigma).
1 g of 0.1 M borate buffer (pH 8.3) 10 ml
Dissolve in water and stir well, then 40 at 40,000g at 4 ℃.
It was prepared by diluting the supernatant after centrifugation for 0.1 minutes with 0.1 M borate buffer.

Also, instead of after the enzymatic reaction, hydrochloric acid was added before the enzymatic reaction and the same operation was performed to prepare a blank. The inhibition rate was calculated using the following formula 1.

[0015]

## EQU1 ## Inhibition rate = {1- (BD) ÷ (AC)} × 100 (%) A: Absorbance when distilled water is added (228 nm) B: Absorbance when inhibitor is added (228 nm) C : Absorbance blank at the time of adding distilled water (228 nm) D: Absorbance blank at the time of adding inhibitor (228 nm)

According to this method, the above-mentioned peptide is Gly-Hi.
Angiotensin I at a concentration of s-Phe of 1100 μM
Inhibits the activity of converting enzyme by 50%, Tyr-Arg-Pr
o-Tyr inhibited by 50% at 380 μM.

HPLC condition 1 Column: RP-18 (e) 1.0 × 25 cm (manufactured by Merck) Mobile phase: A 0.05% trifluoroacetic acid solution B 0.05% trifluoroacetic acid, 30% acetonitrile solution The gradient condition is a linear gradient from 100% of solution A to 100% of solution B after 120 minutes Flow rate: 4.0 ml / min Detection: 210 nm

HPLC condition 2 column: RP-18 (e) 0.46 × 25 cm
(Merck) Mobile phase: A 0.05% trifluoroacetic acid solution B 0.05% trifluoroacetic acid, 30% acetonitrile solution Gradient conditions are linear gradient from 100% of solution A to 100% of solution B after 120 minutes Flow velocity : 1.0 ml / min Detection: 210 nm

HPLC condition 3 Column: GS-220 0.76 × 50 cm (manufactured by Asahi Kasei Corporation) Mobile phase: 50 mM ammonium acetate Flow rate: 0.5 ml / min Detection: 210 nm

Example 2 Boc-Gly (Boc represents a tertiary butoxycarbonyl group), Boc-His (Tos) (Tos represents a tosyl group), Boc-Phe, Boc-Arg
(Tos), Boc-Pro, Boc-Tyr (Bz
L) (Bzl represents a benzyl group) and Boc-Ala using Applied Biosystems Model 430A.
Gly-His-Ph by peptide synthesizer
e, a peptide having the structure of Tyr-Arg-Pro-Tyr was synthesized. The synthesized peptide is YM.
Purification by an MC-ODS-R5 column at a flow rate of 1.0 ml with a linear gradient from 100% of 0.1% trifluoroacetic acid solution to 100% of 70% acetonitrile solution containing 0.1% trifluoroacetic acid after 50 minutes. .. In addition,
The detection was performed by absorption at a wavelength of 210 nm. Tyr-Arg-Pro-Tyr, Gl synthesized and purified in this way
The y-His-Phe peptide was 380 μm each.
M at a concentration of 1100 μM inhibited angiotensin I converting enzyme by 50%.

[0021]

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 obtained at low cost from the unused internal organs of fish and shellfish.

[Sequence list]

SEQ ID NO: 1 Sequence length: 3 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin: Organism name Katsuwonas pelamis
amis)

SEQ ID NO: 2 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin: Biological name Katsuwonas pelamis
amis)

[Brief description of drawings]

FIG. 1 shows a chart separated under HPLC condition 1 in Example 1.

FIG. 2 shows a chart in which peak 1 in FIG. 1 is separated under HPLC condition 2.

FIG. 3 shows a chart in which peak 2 in FIG. 2 is separated under HPLC condition 3.

Claims (3)

[Claims] 1. Gly-His-Phe, Tyr-Ar
An angiotensin I converting enzyme inhibitory peptide having a structure of g-Pro-Tyr. However, Gly is glycine, Hi
s is histidine, Phe is phenylalanine, Tyr is tyrosine, Arg is arginine, and Pro is proline. 2. The method for producing an angiotensin I converting enzyme inhibitory peptide according to claim 1, wherein the seafood is autolysed and purified. 3. The method for producing an angiotensin I converting enzyme inhibitory peptide according to claim 1, wherein the peptide is synthesized by a chemical synthesis method.