JPH04247099A - Angiotensin i converting enzyme-inhibitory tri and tetrapeptide and production thereof - Google Patents

Abstract

PURPOSE:To provide the title peptide capable of inhibiting angiotensin I converting enzyme leading to blood pressure reduction. CONSTITUTION:A peptide extracted from bonito's intestine and purified using the reversed-phase ion exchange chromatography is put to enzymolysis followed by chromatographic separation, thus obtaining the objective peptide having angiotensin I converting enzyme-inhibitory activity of formula Ser-Val-Ala-Lys or Leu-Glu-Lys. For the peptide of the formula Ser-Val-Ala-Lys, IC50=1000muM; for another peptide of the formula Leu-Glu-Lys, IC50=800muM.

Description

Description: [0001] 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 I converting enzyme, which is an enzyme that converts angiotensin I from angiotensin II, which has the effect of increasing blood pressure. [Prior Art] Hypertension is defined as a systolic blood pressure of 160 mmHg.
or above, the diastolic blood pressure is 95 mmHg or above. 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 drugs 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 renal glomerular cells (JG cells) and acts on angiothesinogen, a renin substrate, at blood pressure to produce angiotensin I. The enzyme that converts angiotensin I to angiotensin II is angiotensin I converting enzyme, and the resulting angiotensin II is
It acts on arterioles and causes them to constrict. [0005] Angiotensin II also acts on the adrenal cortex to promote the synthesis and secretion of aldosterone, promotes the reabsorption of Na in the kidney, 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
Decomposed by I. Kininase II is known to be the same substance as angiotensin I exchange 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, zein, gelatin, fish meat, etc. are known to have the effect of inhibiting angiotensin I-converting enzyme (JP-A-59-44324, JP-A-64-
(See No. 5497). [0008] However, although captopril has a strong antihypertensive 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. In addition, S present in the molecule
Because of the H group, it is said to cause rashes and taste abnormalities. Peptides derived from casein, zein, gelatin, fish meat, etc. are natural raw materials, and the manufacturing process generates a lot of unused material, which not only costs a huge amount of money to dispose of, but also wastes money due to disposal. There are concerns about environmental pollution. [0009]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] The present inventors have completed various studies to solve the above problems, and as a result, 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. [0011] The present peptide can be obtained by further enzymatically decomposing the peptide, which is obtained by extracting water from the internal organs of fish and shellfish, heating it, and separating the extracted peptide-containing substance using various chromatography techniques. [0012] This peptide was prepared 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 adjusting the pH to 6.0 and 7.0.
, 8.0, 9.0, the angiotensin I-converting enzyme inhibiting fraction was further subjected to reverse phase HPLC.
It is obtained by separating by LC, enzymatically decomposing the active ingredient, and then separating by reverse phase HPLC. [0013] This peptide is part of tropomyosin, which is widely distributed in the animal body. Therefore, it is possible to extract and isolate tropomyosin from all animals in which it exists. [0014] Furthermore, the present peptide is an Fmoc (9-fluorenylmethyloxycarbonyl)-amino acid.
Fmoc-lysine, Fmoc-glutamic acid, Fmoc
-leucine, Fmoc-alanine, Fmoc-valine,
It can also be produced by solid phase synthesis using Fmoc-serine. [Example] The present invention will be specifically explained below with reference to Examples. Example 1 Add 4 times the amount of water to 17.9 g of bonito intestines, and use a homogenizer (Nissei AM-1 model) to incubate the intestine for 10,000 g on ice.
Homogenize for 5 minutes at 0 rpm. After autoclaving at 120℃ for 5 minutes, autoclaving at 40,000G for 40 minutes4
After centrifugation at ℃ and concentration of the supernatant, C18 packing material (
Waters Bulk C1855-105μm
) The peptide was adsorbed through a simple column packed with 12.4 g. After washing this column with 300 ml of distilled water, the peptide was eluted with a 15% acetonitrile solution. This operation was repeated to obtain 349.6 mg of peptide fraction. [0016] This peptide fraction was prepared using Accel plus equilibrated with 10mM phosphate buffer (pH 6.0).
It was added to a simple column packed with 15 g of CM (Waters), and 10 mM phosphate buffer (pH 6.0) was added.
50ml, (pH 7.0) 250ml, (pH 8.0)
250 ml, (pH 9.0) eluted with 250 ml. Among these, in order to further subdivide the fraction with strong angiotensin I-converting enzyme inhibitory ability, separation was performed by reverse phase HPLC. HPLC conditions are shown in HPLC conditions (■). HPLC conditions ■ Column RP-18(e) 1 x 25 cm
Merck detection UV 230nm Flow rate 4ml/min Eluent A: 0.1% TFAB: 0.1%
TFA 80% acetonitrile 0018 Conditions ■
In this case, the peptide was eluted at a retention time of 16 minutes to 48 minutes. Of these, the eluate with a retention time of 28 to 36 minutes was fractionated and further fractionated by HPLC. The conditions at this time are shown in HPLC conditions (2). HPLC conditions■ Column RP-18(e) 4mm×25c
m Merck detection UV 210nm Flow rate 1.0ml/min Eluent
A: 0.05% TFAB: 0.05% TFA
50% acetonitrile gradient conditions A100
%, A50%, B50% after 800min from B0%
A linear gradient of: ##STR1## Under condition (2), peptide elution was observed at a retention time of 90 to 210 min. Of these, retention time is 90-21
The fraction eluted at 0 min was collected and subjected to HPLC
Further separation was carried out under condition (■). HPLC conditions ■ Column RP-18(e) 4mm x 25c
m Merck detection UV 210nm Flow rate 1.0ml/min Eluent
A: 0.05% TFAB: 0.05% TFA
25% acetonitrile gradient conditions A100
%, A50%, B50% after 800min from B0%
Linear gradient that becomes 0022 HPLC conditions ■
The separated chart is shown in Chart 1. Peak 1~
As a result of measuring the angiotensin I converting enzyme inhibitory ability of No. 14, peak 14 was found to have inhibitory ability. [0023] This peak was applied using Applied Biosys.
stem's gas phase protein sequencer (Mod
el-470A) and online high-performance liquid chromatography to perform the Ddoman decomposition reaction,
The PTH-amino acids obtained in each cycle were identified. As a result, it was estimated to have the structure of SEQ ID NO:1. [0024] Furthermore, JEOL JMS-HX110/
When the FAB-MS spectrum was measured using an HX110 mass spectrometer, ions with a mass number corresponding to a molecular weight of 774 were observed, indicating that the component of peak 14 was the amino acid sequence of SEQ ID NO: 1, as shown above. confirmed. Furthermore, trypsin digestion was performed in a 0.2M ammonium acetate buffer using this peptide as a substrate under enzymatic digestion conditions (2). [0025] Enzyme degradation conditions ■ Substrate concentration 0.1% Enzyme concentration 2% of the substrate Reaction temperature 37°C pH 8.0 Reaction time 24 hours [0026] The enzymatic degradation product was Centricon-10 (A
Trypsin was removed by UF separation using HPLC (Micon), and separation was performed under HPLC conditions (■). The separation pattern is shown in Chart 2. Each peak was identified by determining the amino acid composition ratio using an amino acid analyzer (JEOL model JLC-300). As a result, peak 1 is Ser: Val
:Ala:Lys=1:1:1:1, and it was found to be SEQ ID NO:2. Similarly, peak 2 is Leu
-Glu-Lys=1:1:1, and Leu-G
It turned out to be lu-Lys. As a result of measuring the angiotensin I converting enzyme inhibitory ability, Ser-Val-Ala-Lys had an IC5
0 = 1000 μM, Leu-Glu-Lys is IC
50=800 μM. Angiotensin I-converting enzyme inhibition was measured by the method of Cushman et al.
971)] was improved by the method of Maruyama et al. (Agricultural Biological Chemistry Vol. 46, No. 5 [139
3-1394 (1982)]). That is, 30 μl of an aqueous solution of the peptide-containing composition of the present invention and 250 μl of a pH 8.3 boric acid buffer containing L-hypril histidyl leucine (manufactured by Sigma) and NaCl as enzyme substrates were added to a test tube. 37
Pre-incubated for 10 minutes at °C. 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, and the concentration of L-hipryl histidylleucine was 5M.
M, NaCl is 300 mM, and the enzyme activity without inhibition is 8 mU. 30 at 37°C, pH 8.3
After reacting for a minute with shaking, add 1N HCl 2
50 μl was added to stop the reaction. ethyl acetate 1.5
ml was added and shaken for 15 seconds to extract hippuric acid produced by the enzyme reaction, and centrifuged at 2000 rpm for 10 minutes to collect 1.0 ml of ethyl acetate layer 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 in the enzyme reaction was prepared by dissolving 1 g of Rabbit Lang Acetone Powder (manufactured by Sigma) in 10 ml of 0.1 M boric acid buffer (pH 8.3), stirring well, and incubating at 4°C and 40,000 G for 40 min. A supernatant obtained by centrifugation for one minute was diluted with 0.1 M borate buffer (pH 8.3). The inhibition rate was determined using the following formula. A: Absorbance of distilled water (228 nm) instead of inhibitor B
: Absorbance upon addition of inhibitor (228 nm) Example 2 The peptide of the present invention is Fmoc (9-fluorenylmethyloxycarbonyl)-amino acid (Fmoc-lysine, Fmoc-lysine, Fmoc-lysine,
moc-glutamic acid, Fmoc-leucine, Fmoc
-alanine, Fmoc-valine, Fmoc-serine) by solid phase peptide synthesis. This peptide is
Analysis was performed using a YMC-ODS-R5 column (manufactured by YMC Co., Ltd.) using a 25-minute linear gradient from an initial solution of 0.1% TFA (trifluoroacetic acid) aqueous solution to a final solution of 70% acetonitrile solution of 0.1% TFA. (Flow rate 1.0ml/min, detection UV 210nm)
The results showed a purity of 98%. I is the concentration that inhibits ACE by 50% using this substance.
As a result of measuring the C50 value, Ser-Val-Ala-
Lys is 1000μM, Leu-Glu-Lys is 7
The concentration was 90 μM, which was similar to that produced by enzymatic digestion. [0031] As explained above, according to the present invention,
Angiotensin I-converting enzyme-inhibiting peptides that have the effect of suppressing increases in blood pressure can be obtained from fish and shellfish viscera, which have traditionally been discarded. [Sequence Listing] Sequence number: 1 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Originating organism name: Katsuonas pelamis (Katsuw)
onus pelamis) SEQ ID NO: 2 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Originating organism name: Katsuonus pelamis
onus pelamis)

[Brief explanation of the drawing]

FIG. 1 shows a chart in which fractions with strong angiotensin I-converting enzyme inhibitory ability were separated by HPLC ■ in Example 1.

FIG. 2 shows a chart in which the peptides separated in FIG. 1 were separated by enzymatic degradation.

Claims (5)

[Claims] 1. An angiotensin I converting enzyme inhibitory peptide having the structure Ser-Val-Ala-Lys. (However, Ser is serine, Val is valine, Al
a represents alanine and Lys represents lysine residue. ) 2. An angiotensin I converting enzyme inhibitory peptide having a structure of Leu-Glu-Lys. (Lys indicates lysine, Leu indicates leucine, and Glu indicates glutamic acid residue.) 3. Ser-Val-Ala-Lys, which is characterized by separating and purifying an extract obtained by extracting tropomyosin from an animal organ using a solvent.
3. The method for producing an angiotensin I converting enzyme inhibiting peptide according to claim 1 or 2, which is obtained by enzymatically decomposing an angiotensin I converting enzyme inhibiting peptide having the structure -Leu-Glu-Lys. 4. Ser-Val-Ala-L, which is characterized by solid phase synthesis using Fmoc-lysine, Fmoc-alanine, Fmoc-valine, and Fmoc-serine.
A method for producing an angiotensin I converting enzyme inhibitory peptide having the structure ys. 5. A method for producing an angiotensin I converting enzyme-inhibiting peptide having a Leu-Glu-Lys structure, which comprises solid-phase synthesis using Fmoc-lysine, Fmoc-glutamic acid, and Fmoc-leucine.