JPH04282398A - Angiotensin converting enzyme inhibitor peptide - Google Patents

Abstract

PURPOSE:To obtain the title peptide useful for a medicine, a healthy food, etc., effective for remedying and preventing hypertension, showing inhibitory action on angiotensin converting enzyme, having an amino acid sequence of L-phenylalanin-L-aspartic acid-L-lysine. CONSTITUTION:Cheese whey powder is dissolved in distilled water, adjusted to pH8.0 with sodium hydroxide, mixed with calcium chloride so as to give 5mM final concentration, the prepared solution is warmly kept at 37 deg.C, digested with trypsin for 24 hours while stirring, then the digested solution is adjusted to pH4.5 with hydrochloric acid, unreacted protein and trypsin are precipitated, the precipitate is removed by centrifugal separation, ethanol is added to the solution, protein is further precipitated, supernatant liquid is separated, ethanol is distilled away under reduced pressure, the remaining aqueous solution is successively subjected to gel filtration and high performance liquid chromatography and purified to give the objective peptide containing an amino acid sequence comprising L-phenylalanin-L-aspartic acid-L-lysine.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a novel peptide that has angiotensin converting enzyme (hereinafter referred to as ACE) inhibitory activity and is useful for the treatment or prevention of hypertension.

0002

BACKGROUND OF THE INVENTION Many studies have revealed that the renin-angiotensin system has important significance in the pathology of circulatory system diseases such as hypertension and heart failure. ACE is present in the renin-angiotensin system, and this enzyme is closely related to the regulation of blood pressure. That is, ACE excises a dipeptide from the carboxyl terminal of oligopeptide angiotensin I, which is an active precursor, to generate angiotensin II, which has vasoconstrictive properties. It also has the action of decomposing antihypertensive peptides or bradykinin, and is an enzyme that acts on increases in blood pressure. Therefore, by inhibiting the production of angiotensin II, that is, by inhibiting the activity of ACE, the increase in blood pressure can be suppressed. From the above, natural products,
Synthetic compounds have been searched for ACE inhibitors, and various inhibitors including captopril have been developed.

[0003] As mentioned above, ACE is an aminopeptidase that excises dipeptides, so a peptide that has an affinity for the binding site of ACE can basically become an inhibitor of ACE. Traditionally, it was thought that proteins were broken down into amino acids by the action of various digestive enzymes and absorbed as mere nutrients, but recently there have been reports that some proteins are absorbed from the small intestine as oligopeptides. It is highly likely that the peptide exerts a hormone-like effect on living organisms. Against this background, searches for bioactive peptides derived from foods are being conducted in various fields. Peptides that have immunostimulatory effects are already known, and peptides that inhibit ACE include casein,
It is known to exist in enzymatically degraded products such as soybeans.

0004

SUMMARY OF THE INVENTION An object of the present invention is to provide a peptide that has excellent ACE inhibitory activity and is useful for treating or preventing hypertension.

[0005]

According to the present invention, a peptide containing an amino acid sequence consisting of L-phenylalanine-L-aspartic acid-L-lysine and having ACE inhibitory activity is provided.

The present invention will be explained in more detail below.

[0007] The present invention has ACE inhibitory activity and L
- A peptide containing an amino acid sequence consisting of phenylalanine, L-aspartic acid, and L-lysine, to which a pharmaceutically acceptable salt such as hydrochloride, sulfate, succinate, citrate, or tartrate is added. etc. may be used.

The peptides of the present invention can be prepared, for example, by tryptic decomposition of whey proteins or by peptide synthesis. Specifically, in the case of decomposing whey proteins with trypsin, for example, whey proteins such as cheese whey and acid whey are decomposed with trypsin for 12 to 48 hours under conditions of pH 6 to 9 and temperature of 25 to 50°C, and then decomposed. Trypsin and undegraded proteins are precipitated and removed by adding acid to the sample to obtain a supernatant, and ethanol etc. are added to the supernatant to precipitate the proteins. After removing the ethanol, Sephadex LH-20, Sephadex This can be carried out by separation and purification methods such as gel filtration using Dex G-10, high performance liquid chromatography (HPLC), and the like.

[0009] Furthermore, when using the peptide synthesis method,
According to a known method, for example, the amino group of one amino acid of a peptide is protected with an Fmoc group, the carboxyl group of the other amino acid or peptide is protected with an ester, and coupled with carbodiimide, etc., this operation is repeated, and finally the protecting group is removed. This can be carried out by a method of removing and purifying.

[0010] The peptide of the present invention exhibits a strong inhibitory activity against ACE and has a blood pressure lowering effect, and therefore can be used by being added to, for example, medicines, health foods, or functional foods. Further, the administration form may be oral administration, intravenous administration, etc., and the intake amount varies depending on the subject to whom it is administered, but for example, when administering to humans, 1 to 10
A range of 00 mg/day/kg is preferred. Furthermore, since the peptide of the present invention is non-toxic, it can be administered continuously. For example, when added to health foods, excellent effects can be expected by ingesting it every day. can.

[0011]

Effects of the Invention The peptide of the present invention has excellent ACE inhibitory activity and is excellent in safety, so it can be used in medicines, health foods, functional foods, etc. to treat or prevent hypertension, etc. Excellent effects can be expected.

0012

EXAMPLES The present invention will be explained in more detail below with reference to Examples and Test Examples, but the present invention is not limited thereto.

[0013]

[Example 1] 100g of cheese whey powder and 1 part of distilled water
After adjusting the pH to 8.0 with sodium hydroxide, calcium chloride was added to a final concentration of 5 mM. The resulting solution was kept warm at 37°C and treated with trypsin (
Manufactured by Sigma Chemical Co., TPCK treated, derived from bovine pancreas, 122
00 BAEE unit/mg) was added,
Digestion was carried out for 24 hours with stirring. Next, hydrochloric acid was added to the digestive fluid to adjust the pH to 4.5 to precipitate unreacted proteins and trypsin, followed by centrifugation (8000 rpm).
m, 30 min) to remove the precipitate, and diluted with ethanol (final concentration 8
0%) was added to further precipitate the protein. Ethanol was removed from the obtained supernatant by vacuum concentration, and the aqueous solution was passed through a column of Sephadex LH-20 and purified by elution with distilled water.
Fractions of ~300 ml were collected and concentrated under reduced pressure, and the concentrate was further passed through Sephadex G-10 and eluted with distilled water. Next, the fractions showing the maximum activity shown in FIG.
50 mg was obtained. A small amount of 0.01 wt% TF
A was dissolved in water and separated using an HPLC column, and a fraction showing inhibitory activity was collected.

[0014] The obtained sample was dissolved in 6N hydrochloric acid, heated under vacuum at 110°C for 22 hours, and then analyzed using an amino acid analyzer.
They were contained in a ratio of 1.06 mol he and 1.02 mol L-Lys. In addition, the primary sequence of the obtained peptide was determined using a gas phase protein sequencer PSQ-1 (manufactured by Shimadzu Corporation).
It was found that it has an L-Phe-L-Asp-L-Lys structure.

The column processing conditions for Sephadex LH-20, Sephadex LH-10, and HPLC described above are shown below.

Sephadex LH-20 column processing conditions Column: height 40 cm, inner diameter 4 cm, flow rate: 0.4 ml/min, sample addition amount: 10 ml. Processing conditions for Sephadex LH-10 column Column: Height: 110 cm Internal diameter: 3.5 cm, Flow rate: 10 ml/hour Sample addition amount: 10 ml, Fraction volume: 6 ml. H
PLC column processing conditions HPLC column: Product name “R-ODS-5”, 4.6×
250 mm (manufactured by YMC), A buffer: 0.01% by weight TFA water, B buffer: acetonitrile Flow rate: 1 ml/min. The linear gradient pattern of B buffer from 10 to 60 minutes is shown in Figure 2.

[0017]

[Example 2] Fmoc (9-fluorenylmethoxycarbonyl group)-Lys (Boc) (tert-butyloxycarbonyl)-O-polymer 0.5 g (0.175
mol) was washed three times with 6 ml of dimethylformamide (DMF). The Fmoc group was then deprotected with 20 wt% piperidine/DMF, and DMF, N-methyl-2-
Washed with pyrrolidone (NMP). Then 1-hydroxybenzole hydrate (HOBT) 0.1
g and 1,3-diisopropylcarbodiimide (DIP
Fmo in 6 ml of NMP solution containing 0.09 g of CI)
After performing a coupling reaction with 0.288 g of c-Asp(OBut) (tertiary butyl ester) at room temperature for 2 hours, the excess Fmo was washed 3 times with 6 ml of NMP.
Remove c-Asp(OBut) and place Fmoc-Asp(OBut)-Lys(Boc) on the styrene polymer.
was synthesized. Then, after deprotecting the Fmoc group with 20 wt% piperidine/DMF, 0.1 g of HOBT,
In 6 ml of NMP solution containing 0.09 g of DIPCI,
A coupling reaction was carried out with 0.271 g of Fmoc-Phe at room temperature for 2 hours. After washing with 6 ml of NMP,
Further wash 3 times with 6 ml of DMF to remove excess Fmoc-P.
Remove he and place Fmoc-Phe on styrene polymer.
-Asp(OBut)-Lys(Boc) was synthesized. Further, the Fmoc group was deprotected with 20% by weight piperidine/DMF, and then washed with 6 ml each of DMF, NMP, and methanol in this order. After completely drying the polymer, trifluoroacetate (TFA)-phenol (9
The OBut group and Boc group were deprotected using 5 ml of the 5:5) solution, and the peptide was excised from the polymer. Then add 1-2 m of TFA-phenol (95:5) solution.
The peptide was precipitated by adding a small amount of anhydrous ether. The obtained peptide was dissolved in a small amount of water and purified by Sephadex G-15 to obtain 40 mg of peptide. In addition, amino acid analysis of the obtained peptide revealed that L-Phe0 per mol of L-Asp.
．． 939 mol, L-Lys 1.13 mol.

[0018]

[Test Example 1] After putting 0.04 ml of each of the peptides prepared in Example 1 and Example 2 into test tubes, add 0.1 M borate buffer (0.3 M, containing NaCl, pH 8.3).
Add 0.2 ml of enzyme substrate (Hiplihistidylleucine, manufactured by Sigma Chemical Co., Ltd.) whose final concentration was adjusted to 5 mM using
The mixture was kept warm at ℃ for 10 minutes. Then, add distilled water to 25
0.04 ml of rabbit lung ACE (manufactured by Sigma Chemical Co., Ltd.) adjusted to mU/ml was added and reacted at 37° C. for 30 minutes. Then, 0.25 ml of 1N hydrochloric acid was added to terminate the reaction, and 1.7 ml of ethyl acetate was added.
The mixture was vigorously stirred for 0 seconds, centrifuged at 3000 rpm for 10 minutes, and 1.4 ml of the ethyl acetate layer was collected. After heating the obtained ethyl acetate layer at 120°C for 40 minutes to remove the solvent, 1.0 ml of distilled water was added, and the absorption of the extracted hypolytic acid (absorbance at 228 nm) was measured, and this was determined as the enzyme activity. did.

From the following formula, the inhibitory activity of the peptide IC50 [
Sample concentration required to inhibit ACE activity by 50% (
μg/ml)], it was found that both the peptides prepared in Example 1 and Example 2 had a peptide inhibitory activity IC of
50 was 160 μg/ml. Inhibition rate = (A-B)/(A-C) x 100% A: Sample (
Enzyme activity (absorbance at 228 nm) without peptide) B: Enzyme activity (absorbance at 228 nm) with sample addition
C: Enzyme activity when no enzyme or sample is added (22
absorbance at 8 nm)

[0020]

[Test Example 2] 1 reared with free access to water and feed
A sample (the peptide prepared in Example 1 dissolved in physiological saline) was administered to 2-week-old male spontaneously hypertensive rats (SHR) (Japan Charles River Co., Ltd., 5 rats per group) using a gastric tube. The drug was forcibly administered orally, and blood pressure was measured 6 hours later. The dose of the sample was 50 mg and 100 mg per 1 kg of rat body weight. In addition, for blood pressure measurement, a non-invasive blood pressure measuring device (product name "PE-300", N
ARCO BIO-SYSTEM)
Systolic blood pressure was measured by the l-cuff method. The results are shown in Table 1 as the average value of 5 animals per group.

[0021]

[Table 1]

[Brief explanation of the drawing]

FIG. 1 is a graph showing the elution pattern when the active fraction obtained with Sephadex LH-20 was subjected to gel filtration with Sephadex G-10 in the production process of the peptide of the present invention in Example 1. .

FIG. 2 is a graph showing the results of HPLC analysis of the purified ACE-inhibiting peptide of the present invention in Example 1.

Claims (1)

[Claims] 1. A peptide containing an amino acid sequence consisting of L-phenylalanine-L-aspartic acid-L-lysine and having angiotensin converting enzyme inhibitory activity.