JPH04149196A - Peptide of inhibiting angiotensin i converting enzyme and its production - Google Patents

Abstract

NEW MATERIAL:A peptide of inhibiting angiotensin I converting enzyme, having structure shown by the formula (Ser is serine residue; Val is valine residue; Lys is lysine residue; Leu is leucine residue; Glu is glutamic acid residue). USE:A therapeutic agent of hypertension. PREPARATION:For example, intestine of bonito as a tropomyosin-existing animal organ is mixed with water, homogenized under chilling, treated in an autoclave at 120 deg.C for 5 minutes and centrifuged, the supernatant liquid thereof is concentrated, passed through an adsorption column and the adsorbed substance is treated with 15% acetonitrile solution to elute peptide. The peptide fraction is successively purified by gel filtration or high performance liquid chromatography and a fraction exhibiting strong inhibitary activity against angiotensin I converting enzyme is collected to give a compd. having structure shown by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to an angiotensin I converting enzyme inhibitory peptide and a method for producing the same. More specifically, the present invention relates to a peptide that inhibits angiotensin-converting enzyme, which is an enzyme that converts angiotensin-I from angiotensin-I, which has the effect of increasing blood pressure.

(Prior Art) Hypertension is a condition in which the systolic blood pressure is 160 mmHg or more or the diastolic blood pressure is 95 mmHg or more. It is said that there are approximately 20 million patients in Japan, and it is a disease with a high incidence rate.

Hypertension includes cerebral hemorrhage, cerebral infarction, subarachnoid hemorrhage, angina pectoris,
It is known that various complications occur in a wide range of organs, including myocardial infarction, nephrosclerosis, renal failure, and retinal vein occlusion, and effective therapeutic agents are desired.

One of the mechanisms that regulates blood pressure in vivo is the renin-angiotensin system, which is a pressor system, and the kallikrein-kinin system, which is a blood pressure lowering system. The renin-angiotensin system produces enzymes;
- Angiotensin I is produced by G cells) and acts on angiotensingen, which is a renin substrate in the blood.
generate. The enzyme that converts this angiotensin I to angiotensin H is angiotensin I metabolite, and the produced angiotensin 2 acts on arterioles and causes them to contract.

Angiotensin ■ also acts on the adrenal cortex to promote the synthesis and secretion of aldosterone, promotes the reabsorption of Na in the kidneys, and has the function of retaining body fluids. In this way, angiotensin H increases blood pressure.

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

From the above, by inhibiting angiotensin Ⅰ converting enzyme, the production of angiotensin H, which is a pressor substance, is suppressed, and the decomposition of kinin, which is a hypotensive substance, is suppressed, thereby exhibiting a hypotensive effect. Therefore, treatment of hypertension by inhibiting angiotensin I converting enzyme has been considered.
To date, captopril, enalapril, alacebril, delabril, etc. have been developed. Furthermore, peptides derived from casein, zein, gelatin, fish meat, etc. are known to have the effect of inhibiting angiotensin I-converting enzyme (JP-A-51-44324, JP-A-64
-5497).

However, although Cabtril has a strong blood pressure lowering effect,
It is produced by a synthetic method and is expensive, making it difficult to obtain it 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, zein, gelatin, fish meat, etc. are natural raw materials, and the manufacturing process generates a lot of unused materials, which not only requires enormous costs to dispose of. , there are concerns about environmental pollution due to disposal.

(Problems to be Solved by the Invention) Therefore, it is desired to develop a natural angiotensin I converting enzyme inhibitor that is inexpensively available and has few side effects, and furthermore, to develop an appropriate method for producing the same.

(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 relates to angiotensin I-converting enzyme-inhibiting peptides derived from fish and shellfish internal organs.

This peptide can be obtained by extracting water from the internal organs of seafood, heating it, and then separating the extracted peptide-containing substances using various chromatography techniques.

This peptide was separated by adsorbing the peptide in the aqueous extract onto a reverse phase chromatography packing material Cps (a silica carrier with an octadecyl group bonded to it), eluting it with a 15% acetonitrile solution, and then using an ion exchange chromatography packing material. It is adsorbed on CM (a carrier with a carboxyl methyl group bonded to it), and the pH is adjusted to
Among the fractions obtained by the stepwise elution method of 6,0,7,0°8.0,9.0, the angiotensin I converting enzyme inhibiting fraction is further separated by reverse phase HPLC.

This peptide is part of tropomyosin, which is widely distributed in the animal body. Therefore, it is possible to extract and separate tropomyosin from all animal bodies in which it exists.

In addition, this peptide is an Fmoc (9-fluorenylmethyloxycarbonyl)-amino acid, FsOC-lysine, Fmoc-glutamic acid, Fmoc-leucine,
It can also be produced by solid phase synthesis using Fmoc-alanine, Fmoc-valine, and Fmoc-serine.

(Example) Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1 Four times the amount of water was added to 17.9 g of bonito intestines, and using a homogenizer (Model AM-1), the mixture was cooled on ice for 10. OO
Homogenized for 5 minutes in Orpm.

40,000 after autoclaving at 120°C for 5 minutes
After centrifuging at 4°C for 40 minutes and concentrating the supernatant,
CI8 filler (Waters Bulk Cps 5
The peptide was adsorbed through a simple column packed with 12.4 g of 5-105 μm). Add this column to 300 d of distilled water.
After washing with water, the peptide was eluted with a 15% acetonitrile solution. This operation was repeated to obtain a peptide fraction of 349.6 square meters.

This peptide fraction was prepared using 10mM phosphate buffer (pH 6).
, 0), the accelerator plus CM (Wate
rs) to a simple column packed with 15g of 10mM
Phosphate buffer y-(pH6,0) 250-1 (pH7
,0)250d, (pH8,0)250-1(pH9
,0) At 250d? It eluted.

Of these, in order to further subdivide the two fractions with strong angiotensin J converting enzyme inhibitory ability, separation was performed by reverse phase HPLC. HPLC conditions are shown in HPLC conditions (■).

HPLC conditions ■ Column RP-18(e) I x 25 cm
Merck detection UV 23Or+m Flow rate 4d/l1in Eluent A: 0.1% TFA B: o, 1% TFA 80χ acetonitrile gradient Conditions Under condition (2), the peptide was eluted at a retention time of 16 to 48 minutes. Of these, retention time 28
The eluate from 36 minutes to 36 minutes was fractionated and further analyzed by HPLC.
It was fractionated with The conditions at this time are shown in HPLC conditions (2).

HPLC conditions■ Column RP-18(e) 4 mm x 25
cm Merck detection UV 21On+w flow rate 1. Od/a+in Eluent A: 0.05% TFA B: 0.054 TFA 50χ Acetonitrile Cladding A-+B50χ 800ml
In condition n, the retention time was 90 to 210 gl1
Peptide elution was observed at n. Among these, the fraction eluted at a retention time of 90 to 210 m1n was collected and further separated under HPLC conditions (2).

HPLC conditions■ Column RP-18(e) 4 mm x 25
cv Merck detection UV 21Or+s Flow rate 1.0d/w+in Eluent A: 0.05% TFAB: 0.05
4 To TF8 5χ Acetonitrile A→B 50χ
A chart separated under 800 m1n linear gradient HPLC condition (2) is shown in the drawing. This chart was separated into 14 peaks as shown in the drawing. Therefore, the in vitro ACE inhibition ability of each peak was measured.

Angiotensin I-converting enzyme inhibition was measured by the method of Shirimaruyama et al. (Agricultural Biological Chemistry Vol. 46, 5), which was modified from the method of Cushman et al. No. (1393-1394 pages (19B)
2) According to ).

That is, an aqueous solution 3 of the peptide-containing composition of the present invention is placed in a test tube.
0 .mu.r and 250 .mu.r of boric acid heffer containing L-hypril histidyl leucine (manufactured by Sigma) and NaCl as enzyme substrates at pH 8.3 were added, and preincubation was carried out at 37.degree. C. for 10 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 was 0.1M, L-
Hibril histicilleucine concentration is 5111M, NaC
The enzyme activity is 1300mM and the enzyme activity without inhibition is 8mU. After reacting with vibration at 37°C and pH 8.3 for 30 minutes, IN) IC1250μ! was added to stop the reaction. Add 1.5 g of ethyl acetate,
Extract the hippuric acid produced by the enzymatic reaction by shaking for 20 seconds.
Centrifuge at 0 rpm for 10 minutes to remove ethyl acetate layer 1.0
Males were collected in test tubes. Ethyl acetate was completely removed by heating at 120° C. for 130 minutes in a hot dry path, and then left at room temperature for 5 minutes. And 8201. 0- was added, and the amount of hippuric acid produced was determined by measuring the absorbance at 228r+m. Angiotensin I used for enzyme reaction
The converting enzyme-containing solution is Rabbit Lang Acetone Powder (
(manufactured by Sigma) Ig was 0. IM boric acid Hamfer (pH 8
, 3) Rinse to 10m and stir well, then heat to 4°C840
,000g for 40 minutes and the supernatant was centrifuged at 0.000g for 40 minutes. It was prepared by diluting it with IM borate buffer (pH 8,3).

The inhibition rate was determined using the following method.

As a result of measuring the ACE inhibition rate for beaks ■ to ■ in the chart shown in the drawing, a high inhibition rate was observed in the beak [phase]. This peak was measured using Appliecl Biosyst.
Em's gas phase protein sequencer (Model-
470A) and online high-performance liquid chromatography to perform the Edoman degradation reaction, and identify the PTH-amino acids obtained in each cycle. As a result, Ser
-Sial-Ala-Lys-Leu-Glu-Lys structure.

Furthermore, JEOL JMS-HX 110/HX110
When the FAB-MS spectrum was measured using a type mass spectrometer, ions with a mass number corresponding to a molecular weight of 774 were observed, and as shown above, the component of beak 14 was Ser-Sial-Ala-Lys-Leu- Glu-L
It was confirmed that ys.

This peptide is Fmoc (9-fluorenylmethyloxycarbonyl)-amino acid (Fmoc-lysine, Fsoc
-glutamic acid, Fmoc-leucine, Fmoc-alanine, Fmoc-valine, Fmoc-serine). This peptide is YMC
-0 with ODS-R5 column (manufactured by YUMUSHI Co., Ltd.)
．． Analysis was performed using a 25-minute linear gradient from an initial solution of 1% TFA (refluoroacetic acid) aqueous solution to a final solution of 0.1% TFA in 70% acetonitrile (
Flow rate 1. 0d/sin, detection IJV210nIll)
The results showed a purity of 98%. Using this substance, the IC3° value, which is the concentration that inhibits ACE by 50%, was measured and found to be 82.3μ.

(Effects of the Invention) As explained above, according to the present invention, an angiotensin I-converting enzyme inhibiting peptide having the effect of suppressing an increase in blood pressure can be obtained from conventionally discarded fish and shellfish viscera.

[Brief explanation of drawings]

The figure shows a chart in which the fraction with strong angiotensin I converting enzyme inhibitory ability was further separated under HPLC conditions (2) in Example 1. (1 other person)

Claims (3)

[Claims] (1) Ser-Val-Ala-Lys-Leu-Gl
Angiotensin I converting enzyme inhibitory peptide with the structure of u-Lys. (However, Ser is serine, Val is valine, Ala is alanine, Lys is lysine, Leu is leucine, Glu
indicates a glutamic acid residue. ) (2) Ser-Val-Ala-Lys-Leu-G characterized by separating and purifying an extract obtained by extracting using a solvent from an animal organ in which tropomyosin exists.
A method for producing an angiotensin I converting enzyme inhibitory peptide having a lu-Lys structure. (3) Fmoc-lysine, Fmoc-glutamic acid, F
Ser-Val-Ala-Lys-Leu-Glu- characterized by solid phase synthesis of moc-leucine, Fmoc-alanine, Fmoc-valine, and Fmoc-serine
A method for producing an angiotensin I converting enzyme inhibitory peptide having a Lys structure.