JP3149199B2 - Angiotensin converting enzyme inhibitory peptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel peptide having angiotensin converting enzyme (hereinafter referred to as ACE) inhibitory activity and useful for treating or preventing hypertension.

[0002]

2. Description of the Related Art Many studies have revealed that the renin-angiotensin system has important significance in the pathology of cardiovascular diseases such as hypertension and heart failure.
ACE is present in the renin-angiotensin system, and this enzyme has a close relationship with the regulation of blood pressure. That is, ACE cuts out a dipeptide from the carboxyl terminal of oligopeptide angiotensin I, which is an active precursor, to produce angiotensin II having vasoconstriction. In addition, it has an action of decomposing a hypotensive peptide or bradykinin, and is an enzyme that acts on increasing blood pressure. Thus inhibiting the production of angiotensin II,
That is, by inhibiting the activity of ACE, an increase in blood pressure can be suppressed. From the above, ACE inhibitors have been searched for natural products and synthetic products, and various inhibitors including captopril have been developed.

[0003] Since ACE is an aminopeptidase that cleaves dipeptides as described above, a peptide having an affinity for the binding site of ACE can basically be an ACE inhibitor. Conventionally, it has been thought that proteins are decomposed into amino acids by the action of various digestive enzymes and are simply absorbed as nutrients, but recently it has been reported that some proteins are absorbed as oligopeptides from the small intestine. It is highly possible that the peptide has a hormone-like effect on the living body. Against this background, search for bioactive peptides derived from foods has been carried out in various fields. Peptides having an activity such as immunostimulation are already known, and peptides that inhibit ACE are casein,
It is known to be present in enzymatic degradation products such as soybeans.

[0004]

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

[0005]

According to the present invention, L-phenylalanine-L-aspartic acid-L-lysine is used.
A peptide having an angiotensin converting enzyme inhibitory activity comprising an amino acid sequence represented by the formula:

Hereinafter, the present invention will be described in more detail.

[0007] The present invention provides an ACE inhibitory activity,
-Phenylalanine-L-aspartic acid-L-lysine
And a peptide to which a pharmaceutically acceptable salt such as hydrochloride, sulfate, succinate, citrate, tartrate and the like is added.

The peptide of the present invention can be prepared, for example, by a method of trypsinizing whey protein or a peptide synthesis method. When trypsin-decomposing whey protein specifically, for example, whey protein such as cheese whey, acid whey, pH 6-9,
After trypsin degradation for 12 to 48 hours at a temperature of 25 to 50 ° C., trypsin and undegraded protein are removed by precipitation by adding an acid to the decomposed product to obtain a supernatant, and ethanol and the like are added to the supernatant. After precipitating the protein and then removing ethanol, the separation can be carried out by a method such as gel filtration using Sephadex LH-20 or Sephadex G-10, or separation and purification by high performance liquid chromatography (HPLC).

When using the peptide synthesis method,
According to a known method, for example, the amino group of one amino acid of the peptide is protected with an Fmoc group, the carboxyl group of the other amino acid or the peptide is protected with an ester, coupled with carbodiimide or the like, and this operation is repeated. Can be removed and purified.

[0010] The peptide of the present invention has a strong inhibitory activity against ACE and has a blood pressure lowering effect, and thus can be used by adding it to, for example, pharmaceuticals, health foods or functional foods. The administration form may be any of oral administration, intravenous injection administration, etc., and the amount of intake varies depending on the subject to be administered.
A range of 00 mg / kg / day is preferred. Furthermore, since the peptide of the present invention has no toxicity, it can be continuously administered.For example, when it is used by adding it to health foods or the like, it is expected that excellent effects can be expected by taking it daily. it can.

[0011]

Industrial Applicability The peptide of the present invention has excellent ACE inhibitory activity and excellent safety, so that it can be used in medicine, health food or functional food to treat or prevent hypertension, etc. An excellent effect can be expected.

[0012]

The present invention will be described in more detail with reference to examples and test examples, but the present invention is not limited to these examples.

[0013]

Example 1 100 g of cheese whey powder was added to distilled water 1
After dissolving in 1 liter and adjusting the pH to 8.0 with sodium hydroxide, calcium chloride was added to a final concentration of 5 mM. The obtained solution was kept at 37 ° C., and trypsin (manufactured by Sigma Chemical Co., TPCK treatment, bovine pancreas-derived, 12
(200 BAEE unit / mg) was added and digestion was performed for 24 hours with stirring. Next, hydrochloric acid was added to the digested solution to adjust the pH to 4.5, and unreacted proteins and trypsin were precipitated.
Min), the precipitate was removed, and ethanol (final concentration 80%) was added to further precipitate the protein. Ethanol was removed from the obtained supernatant by concentration under reduced pressure, passed through a column of Sephadex LH-20 as an aqueous solution, and eluted with distilled water for purification.
The fractions were collected and concentrated under reduced pressure. The concentrate was further passed through Sephadex G-10 and eluted with distilled water. Next, the fraction showing the maximum activity shown in FIG. 1 (fraction N
o. 55-60) were collected and lyophilized to give a white powder of 150 m
g was obtained. A part of this was dissolved in a small amount of 0.01% by weight TFA water and separated by an HPLC column, and a fraction showing inhibitory activity was collected.

The obtained sample was dissolved in 6N hydrochloric acid, heated at 110 ° C. under vacuum for 22 hours, and analyzed by an amino acid analyzer. As a result, 1 mol of L-Asp
The content was 1.06 mol of he and 1.02 mol of L-Lys. The primary sequence of the peptide was determined by using a gas-phase protein sequencer PSQ-1 (manufactured by Shimadzu Corporation).
It was found to have an L-Phe-L-Asp-L-Lys structure.

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

Column processing conditions of Sephadex LH-20 Column: height 40 cm, inner diameter 4 cm, flow rate: 0.4 ml
/ Min, sample addition amount: 10 ml. Processing conditions of Sephadex LH-10 column Column: height, 110 cm inner diameter 3.5 cm, flow rate: 10
ml / hour Sample addition amount: 10 ml, fraction volume: 6 ml. HPLC column processing conditions HPLC column: trade 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 the B buffer for 10 to 60 minutes is shown in FIG.

[0017]

Example 2 Fmoc (9-fluorenylmethoxycarbonyl group) -Lys (Boc) (tert-butyloxycarbonyl)
0.5 g (0.175 mol) of -O-polymer was washed three times with 6 ml of dimethylformamide (DMF). The Fmoc group was then deprotected with 20% by weight piperidine / DMF,
Washing was performed in the order of MF and N-methyl-2-pyrrolidone (NMP). NMP containing 0.1 g of 1-hydroxyl benzol hydrate (HOBT) and 0.09 g of 1,3-diisopropylcarbodiimide (DIPCI)
After performing a coupling reaction with 0.288 g of Fmoc-Asp (OBut) (tertiary butyl ester) in 6 ml of the solution at room temperature for 2 hours, the solution was washed three times with 6 ml of NMP and excess Fmoc
-Asp (OBut) is removed and Fmoc-Asp
(OBut) -Lys (Boc) was synthesized. Then, after deprotection of the Fmoc group with 20% by weight piperidine / DMF, HO was added.
In 6 ml of an NMP solution containing 0.1 g of BT and 0.09 g of DIPCI, a coupling reaction was performed with 0.271 g of Fmoc-Phe at room temperature for 2 hours. After washing with 6 ml of NMP,
Further washing with 6 ml of DMF three times to remove excess Fmoc-Phe, and Fmoc-Phe-Asp (OBut) -Lys on styrene polymer.
(Boc) was synthesized. 20% by weight piperidine / DMF
After deprotection of the Fmoc group by means of, DMF, NMP and methanol were sequentially washed with 6 ml each. After the polymer has completely dried, trifluoroacetate (TF
A) OBu with 5 ml of a phenol (95: 5) solution
The t- and Boc groups were deprotected and the peptide was cleaved from the polymer. Then, TFA-phenol (9
5: 5) The solution was concentrated to 1-2 ml, and a small amount of anhydrous ether was added to precipitate the peptide. The obtained peptide was dissolved in a small amount of water and purified by Sephadex G-15 to obtain 40 mg of the peptide. When the obtained peptide was subjected to amino acid analysis, L-Asp 1 mol
With respect to L-Phe 0.939 mol, L-Lys
It was contained at a rate of 1.13 mol.

[0018]

Test Example 1 0.04 ml of each of the peptides prepared in Examples 1 and 2 was put into a test tube, and then 0.1 M borate buffer (containing 0.3 M, NaCl, pH 8.3).
Then, 0.2 ml of enzyme substrate (hyprihistidylleucine, manufactured by Sigma Chemical Co.) adjusted to a final concentration of 5 mM with
Incubated at 10 ° C. for 10 minutes. Then, add distilled water to add 25
Rabbit lung ACE (manufactured by Sigma Chemical Co.) 0.04 ml adjusted to mU / ml was added and reacted at 37 ° C. for 30 minutes. Thereafter, 0.25 ml of 1N hydrochloric acid was added to terminate the reaction, and 1.7 ml of ethyl acetate was added thereto.
The mixture was vigorously stirred for 0 seconds, centrifuged at 3000 rpm for 10 minutes, and 1.4 ml of an 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 (absorbance at 228 nm) of the extracted hypoprilic acid was measured. did.

From the following formula, the inhibitory activity IC _{50 of the} peptide [A
The sample concentration required to inhibit the activity of CE by 50% (μ
g / ml)], the results are shown in Examples 1 and 2.
Both of the peptides prepared in the above were used for the inhibitory activity IC _{50 of the} peptide.
Was 160 μg / ml. Inhibition rate = (AB) / (AC) × 100% A: Enzyme activity without sample (peptide) (22
B: Enzyme activity when sample is added (absorbance at 228 nm) C: Enzyme activity when enzyme and sample are not added (22
8 nm absorbance)

[0020]

Test Example 2 Raised with water and feed ad libitum
To a 2-week-old male spontaneously hypertensive rat (SHR) (Charles River Japan, 5 rats per group), a sample (the peptide prepared in Example 1 dissolved in physiological saline) was added to a gastric tube. And the blood pressure was measured 6 hours later. The doses of the samples were 50 mg and 100 mg per 1 kg of rat body weight. In addition, a non-invasive blood pressure measurement device (trade name “PE-300”,
Systolic blood pressure was measured by tail-cuff method using NARCO BIO-SYSTEM). Table 1 shows the results as the average of 5 animals per group.
Shown in

[0021]

[Table 1]

[Brief description of the drawings]

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

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ identification code FI C12P 21/06 A61K 37/64 (58) Investigated field (Int.Cl. ⁷ , DB name) C07K 5/08 A61K 38/55 A23L 1/305 BIOSIS (DIALOG) CA (STN) REGISTRY (STN) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A peptide comprising an amino acid sequence represented by L-phenylalanine-L-aspartic acid-L-lysine and having angiotensin converting enzyme inhibitory activity.