JPH04297496A - Angiotensin i converting enzyme inhibiting hexapeptide and its production - Google Patents

Abstract

PURPOSE:To provide an angiotensin I converting enzyme inhibiting peptide available at a low cost and having low side effect and to provide a process for the proper production of the peptide. CONSTITUTION:The objective angiotensin I conversing enzyme inhibiting peptide has the structure of formula Gly-Val-Tyr-Pro-His-Lys and is separated and purified from the extract of the guts or muscle of fish and shellfish or chemically synthesized by using Fmoc-glycine, Fmoc-valine, Fmoc-tyrosine, Fmoc- proline, Fmoc-histidine and Fmoc-Lysine. Gly-Val-Tyr-Pro-His-Lys has an angiotensin I converting enzyme inhibition activity of IC<50>=1.6muM.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to an angiotensin I converting enzyme inhibitory peptide and a method for producing the same. More specifically, angiotensin II, which has the effect of increasing blood pressure.
The present invention relates to an angiotensin I converting enzyme inhibitory peptide, which is an enzyme that converts angiotensin I, and a method for producing the same.

0002

[Prior Art] Hypertension is characterized by a systolic blood pressure of 160 mmHg.
95 mmHg or higher, or systolic blood pressure is 95 mmHg or higher, or both are higher than that. It is said that there are approximately 20 million patients in Japan, and it is a disease with a high morbidity rate.

Hypertension is known to cause various complications affecting a wide range of organs, including cerebral hemorrhage, cerebral infarction, subarachnoid hemorrhage, angina pectoris, myocardial infarction, nephrosclerosis, renal failure, and retinal vein occlusion. Therefore, effective therapeutic agents are desired.

One of the mechanisms for regulating blood pressure in vivo is the renin-angiotensin system, which is a pressor system, and the kallikrein-kinin system, which is a hypotensive system. In the renin-angiotensin system, the enzyme renin is produced in the glomerular cells (JG cells) of the kidney and acts on angiothesinogen, a renin substrate, in blood vessels to produce angiotensin I. The enzyme that converts angiotensin I to angiotensin II is angiotensin I converting enzyme, and the generated angiotensin II acts on arterioles to cause them to contract.

[0005] Angiotensin II also acts on the adrenal cortex to promote the synthesis and secretion of aldosterone, promotes the reabsorption of sodium in the kidneys, and has the function of retaining body fluid volume. In this way, angiotensin II increases blood pressure.

On the other hand, in the kallikrein-kinin system, kallikrein, a protease, acts on kininogen, a substrate, to produce kinin. Kinin has the effect of dilating blood vessels and lowering blood pressure, but kininase I
undergoes decomposition by I. Kininase II is known to be the same substance as angiotensin I converting enzyme.

[0007] Based on the above, treatment of hypertension by inhibiting angiotensin I converting enzyme has been considered, and to date, captopril, enalapril, alacepril,
Delapril etc. have been developed. In addition, peptides derived from casein, tzein, gelatin, fish meat, etc. are known to have the effect of inhibiting angiotensin I converting enzyme (JP-A-59-44324, JP-A-64-
No. 5497, Japanese Unexamined Patent Publication No. 1-313498).

[0008] However, although captopril has a strong antihypertensive effect, it is produced by a synthetic method, so it is expensive and difficult to obtain easily. Moreover, inappropriate dosage results in renal dysfunction and hypotension. It is also said to cause rashes and taste abnormalities due to the SH group present in the molecule. Peptides derived from casein, tzein, fish meat, etc. are natural raw materials, so they generate a lot of unused material during the manufacturing process, which not only costs a huge amount of money to dispose of, but also causes environmental damage due to disposal. There are concerns about contamination.

[0009]

[Problems to be Solved by the Invention] The present invention provides an angiotensin I-converting enzyme inhibitory peptide that is inexpensively available, has few side effects, and does not generate new unused substances during production, and an appropriate method for producing the same. The purpose is to provide

0010

[Means for Solving the Problems] As a result of various studies to solve the above problems, the present inventors have completed the present invention. That is, the present invention provides Gly-Val-T
It is an angiotensin I converting enzyme inhibitory peptide having a structure of yr-Pro-His-Lys.

[0011] The present invention also provides the above-mentioned angiotensin I
The converting enzyme inhibiting peptide is extracted from the internal organs and muscles of fish and shellfish, or furthermore, Fmoc-glycine, Fmoc-valine, Fmoc-tyrosine, Fmoc-proline, Fm
This is a method for producing by solid phase synthesis using oc-histidine and Fmoc-lysine. This peptide can be obtained by extracting water from the internal organs and muscles of seafood, heating it, and then separating the extracted peptide-containing substances using various chromatography techniques.

[0012] This peptide was obtained by adsorbing the peptide in the aqueous extract onto C18 packing material for reverse phase chromatography (a silica carrier with an octadecyl group bonded to it), and after washing with water, the fraction was eluted with a 15% acetonitrile solution. Weak cation exchanger CM
(carboxymethyl group bound to a carrier), and among the fractions obtained by stepwise elution, the angiotensin I converting enzyme inhibiting fraction was further separated by reverse phase HPLC,
The active fraction can be further separated by gel filtration HPLC.

[0013] Any fish species that can be used in the present invention includes bonito, tuna, horse mackerel, mackerel, sardines, saury, yellowtail, sea bream, flounder, flounder, sea bass, salmon, herring, squid, and octopus. Internal organs include stomach, pylorus,
Any of the intestine, liver, testis, ovary, heart, etc. may be used. Also, seafood muscles can be used.

[0014] The present peptide also contains Fmoc-glycine, Fmoc-valine, Fmoc-tyrosine, Fmoc-
It can also be produced by chemical synthesis methods such as solid phase synthesis using proline, Fmoc-histidine, and Fmoc-lysine.

[0015]

[Examples] The present invention will be specifically explained below with reference to Examples. Example 1 Four times the amount of water was added to 129.4 g of bonito liver, and using a homogenizer (Nissei AM-1 model), the mixture was heated on ice for 10 minutes.
Homogenization was performed for 5 minutes at 000 rpm.

[0016] After autoclaving at 120°C for 5 minutes,
Centrifugation was performed at 40,000G for 40 minutes at 4°C, the supernatant was concentrated, and then packed with C18 packing (Waters Bu
It was adsorbed onto a simple column packed with 90 g of lk C18 (55-105 μm). After washing this column with 1000 ml of distilled water, the peptides were eluted with a 15% acetonitrile solution to obtain a peptide fraction.

This peptide fraction was prepared using Accel CM (Wa) equilibrated with 10mM phosphate buffer (pH 6.0).
Ters Inc.) was added to a simple column packed with 15 g.
200ml of 0mM phosphate buffer (pH 6.0), (
pH 7.0) 200ml, (pH 8.0) 200ml,
(pH 9.0) and eluted with 200 ml. Among these, in order to further purify the pH 9.0 elution fraction, which has a strong angiotensin I converting enzyme inhibiting ability, separation was performed by reverse phase HPLC. The HPLC conditions are shown in HPLC Conditions 1 below.

[0018] HPLC conditions 1 Column RP-18(e) 1 x 25 cm
Merck detection UV 210nm Flow rate 4ml/min Eluent A: 0.05% TFAB:
0.05% TFA 50% Acetonitrile A10
Linear gradient from 0% to A50% and B50% after 800min

Under condition 1, the peptide having the ability to inhibit angiotensin I converting enzyme was eluted at a retention time of 183 minutes. To further purify this peptide, H
Purified by PLC. The conditions at this time are shown in HPLC conditions 2 below. In addition, Figure 1 shows a chart showing the separation of the fraction with strong angiotensin I-converting enzyme inhibitory ability under HPLC condition 2.
Shown below.

[0020] HPLC conditions 2 Column RP-18(e) 4mm x 25cm
Merck detection UV 210nm Flow rate 1.0ml/min Eluent A: 0.05% TFAB:
0.05% TFA 25% Acetonitrile A10
Linear gradient from 0% to A50% and B50% after 800min

Under condition 2, a strong ability to inhibit angiotensin I-converting enzyme was observed at a retention time of 230 minutes (peak 1 in FIG. 1). In order to further purify this peak, HPLC was performed under the HPLC conditions shown in Condition 3 below. Further, a chart in which peak 1 in FIG. 1 was separated under HPLC condition 3 is shown in FIG.

[0022] HPLC conditions 3 Column Asahi Pack GS-220 7.
5mm x 50cm Asahi Kasei Corporation Detection UV 210nm Flow rate 1.0ml/min Eluent 50mM ammonium acetate solution

002
3] Under these conditions, a peptide with angiotensin I converting enzyme inhibitory ability was eluted at a retention time of 18 minutes (peak 2 in FIG. 2). Applied this peak
Edman degradation reaction was performed using a Biosystem gas phase protein sequencer (Model-470A) and online high performance liquid chromatography, and the PTH amino acid obtained in each cycle was identified. As a result, Gly-Val-Tyr-Pro-His-
It was found that it has the structure of Lys.

The inhibition of angiotensin I converting enzyme was measured using the method of Maruyama et al. [Agricultural Biological Chemistry, Vol. Volume 46, No. 5, 1393-139
4 (1982)].

That is, in a test tube, 30 μl of the peptide aqueous solution of the present invention and a pH 8 solution containing L-hypril histidyl leucine (manufactured by Sigma) and NaCl as enzyme substrates were prepared.
．． Add 250 ml of boric acid buffer from step 3 and incubate at 37°C.
Pre-incubated for 0 minutes. Thereafter, 100 μl of an angiotensin I converting enzyme-containing solution was added to start the enzyme reaction. At this time, the concentration of boric acid buffer is 0.1
M, L-hypril histidyl leucine concentration is 5mM, N
The enzyme activity is 8 mU when aCl is 300 mM and no inhibition is applied. After reacting with shaking at 37°C and pH 8.3 for 30 minutes, 1N HCl 25
0 ml was added to stop the reaction. Ethyl acetate 1.5m
1, shaken for 15 seconds to extract hippuric acid produced by the enzyme reaction, centrifuged at 2000 rpm for 10 minutes,
1.0 ml of the ethyl acetate layer was collected into a test tube. Ethyl acetate was completely removed by heating in a hot dry bath at 120° C. for 30 minutes, and then left at room temperature for 5 minutes. and,
1.0 ml of H2O was added, and the amount of hippuric acid produced was determined by measuring absorbance at 228 nm. The angiotensin I converting enzyme-containing solution used for the enzyme reaction was prepared by dissolving 1 g of rabbit acetone powder (manufactured by Sigma) in 10 ml of 0.1 M boric acid buffer (pH 8.3), stirring well, and centrifuging at 40,000 G for 40 minutes at 4°C. The separated supernatant was diluted with 0.1M boric acid buffer.

The inhibition rate was determined using the following formula. A: Absorbance when adding distilled water (228 nm) B:
Absorbance upon Addition of Inhibitor (228 nm) The IC50 (concentration that inhibits enzyme activity by 50%) of the above peptide was measured using this method and found to be 1.6 μM.

Example 2 The peptides of the present invention are Fmoc-(9-fluorenylmethyloxycarbonyl) amino acids (Fmoc-glycine, Fmoc-valine, Fmoc-tyrosine, Fmoc-
proline, Fmoc-histidine, Fmoc-lysine)
It was synthesized using solid phase synthesis method. This peptide is YM
Using a C-ODS-R5 column (manufactured by YMC Co., Ltd.), a 25-minute linear gradient was applied from an initial solution of 0.1% TFA (trifluoroacetic acid) aqueous solution to a final solution of 70% acetonitrile solution of 0.1% TFA. Analysis (flow rate 1.0 ml/min, detection UV 210 nm) showed a purity of 98%. IC using this substance
As a result of measuring 50, the value was the same as that extracted from bonito intestines.

[0028]

According to the present invention, an angiotensin I-converting enzyme-inhibiting peptide having the effect of suppressing blood pressure increase can be obtained from fish and shellfish viscera that have been conventionally discarded.

[Sequence list]

SEQ ID NO: 1 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type:
Peptide origin organism name: Katsuonas pelamis (Katsuw)
onus pelamis) sequence

[Brief explanation of the drawing]

FIG. 1 shows a chart in which a fraction with a strong angiotensin I converting enzyme inhibitory ability was separated under HPLC conditions 2 in Example 1.

FIG. 2 shows a chart in which peak 1 in FIG. 1 was further separated under HPLC conditions 3.

Claims (1)

[Scope of Claims] [Claim 1] Gly-Val-Tyr-Pro-H
Angiotensin I converting enzyme inhibitory peptide having an is-Lys structure. (However, Gly is glycine, Val is valine, Tyr is tyrosine, Pro is proline, His
indicates histidine, and Lys indicates a lysine residue. ) [Claim 2] G characterized by separating and purifying an extract obtained by extracting the internal organs and muscles of seafood using a solvent.
A method for producing an angiotensin I converting enzyme inhibitory peptide having a structure of ly-Val-Tyr-Pro-His-Lys. 3. Fmoc-glycine, Fmoc-valine, Fmoc-tyrosine, Fmoc-proline, Fmo
Gly-Val-Tyr-Pro-, which is characterized by solid phase synthesis using c-histidine and Fmoc-lysine.
A method for producing an angiotensin I converting enzyme inhibitory peptide having a His-Lys structure.