JP3341256B1 - Peptides with antihypertensive activity - Google Patents

Abstract

Abstract: To find an ACE inhibitor derived from a natural product and to clarify its composition. SOLUTION: An attempt was made to isolate an active peptide by chromatography from enzymatic digestion of striated muscle of the Japanese oyster oyster. The amino acid composition and sequence of the obtained ACE inhibitory activity peptide were examined. ACE inhibitory peptides obtained from enzymatically treated striated muscle are Asp (aspartic acid), Leu
(Leucine), Thr (threonine), and Tyr (tyrosine), and the ratio is 2:
1: 1: 1 and the sequence was Asp-Leu-Thr-
Asp-Tyr. This pentapeptide is decomposed by digestive enzymes in the intestinal tract and is converted into a peptide having a smaller molecular weight to act. In this case, Asp-T
yr (DY) has strong degradation resistance and the strongest blood pressure lowering effect.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to natural AC-derived AC
The present invention relates to an E inhibitor, and particularly to a peptide having an antihypertensive effect extracted from oysters.

[0002]

[Technical background] ACE (angiotensin converting enzym)
e: angiotensin converting enzyme) is an enzyme that removes the C-terminal dipeptide of angiotensin I. This enzyme activates the renin-angiotensin system (hypertensive system) and the kallikrein-kinin system (hypertensive system)
Has an inactivating effect at the same time. An ACE inhibitor that inhibits ACE acts on ACE and can be expected to have an effect of suppressing an increase in blood pressure (vasoconstriction).
It is attracting attention from the viewpoint of preventing hypertension. Usually, hypertension is treated with pharmacotherapy, but the drug has some side effects. Therefore, in recent years, detection of ACE inhibitors derived from natural products has been actively performed.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to find an ACE inhibitor derived from a natural product, clarify its composition, and confirm that a compound having the composition has an ACE inhibitory effect. It is.

The present invention, which satisfies the above-mentioned requirements, has an amino acid sequence of Asp-Leu-Thr-As.
It is a peptide having an antihypertensive effect, which is p-Tyr or Asp-Tyr. This peptide is derived from the striated muscle of the oyster, and the synthesized peptide has a remarkable blood pressure lowering effect.

[0004]

Embodiments of the present invention will be described in detail with reference to the drawings. The present invention relates to a peptide extracted from a Japanese oyster (the same or different α-amino acid in which the amino group at one α-position and the other carboxyl group are dehydrated and bonded. ACE inhibitory activity). The inventor of the present invention measured the ACE inhibitory activity of a mixture of peptides obtained by treating various tissues of the Japanese oyster with various proteases (enzymes that hydrolyze peptide bonds of proteins). Then, they searched for the active ingredient and clarified the amino acid sequence and the like.
Further, the searched peptide was used for spontaneously hypertensive rats (SH
R) was orally administered and the effect of lowering blood pressure in an in vivo system was confirmed.

<Comparison of oyster ACE inhibitory activity> First, proteases used for peptide production were screened. For this, trypsin,
ACE inhibitory activity was measured for each peptide mixture obtained by enzymatic degradation treatment with pepsin and various other proteases. The measurement of the ACE inhibitory activity is described in Lieberman
Substrate Hip-Hi according to the method of Yamamoto et al.
s-Leu is expressed as inhibitory activity against ACE derived from rabbit lung. This assay has also been used to measure other ACE inhibitory activities described herein. The result is shown in FIG. As can be seen from FIG. 1, ACE inhibitory activity was observed in all peptide mixtures. Of these, the pepsin-treated product had the strongest inhibitory activity, followed by the trypsin-treated product. Based on this result, using these two enzymes (pepsin, trypsin), using a mixture of peptides obtained from the gill (ella) of the oyster, striated muscle of the putamen muscle, hemolymph, gonad, and mantle, The inhibitory activity of each tissue was measured. The results are shown in FIG. 2 and FIG. FIG.
Shows the ACE inhibitory activity of each of the tissues of the oysters treated with pepsin. FIG. 3 shows the ACE inhibitory activity of each tissue of the Japanese oysters treated with trypsin. As can be seen from these figures, a strong activity was observed in striated muscle and gonads from a comparison of the inhibitory activities of the peptide mixtures obtained from each tissue.

<Confirmation of antihypertensive effect in vivo> In order to confirm the antihypertensive action of the oyster-peptide mixture in an in vivo system, an administration test was performed on spontaneously hypertensive rats (SHR). The striated muscle muscle was decomposed with trypsin, and the dialysate (fraction molecular weight: about 10,000) was used as the administration sample. As test plots, 50 mg and 100 mg p
After establishing an eptide / kg administration group and orally administering with a probe, the blood pressure was measured using a non-invasive tail arterial blood pressure measurement device.
It was measured over time by the uff method. In addition, the same experiment was performed on pepsin hydrolyzate of the oyster gonad. The results are shown in FIGS. FIG. 4 shows the time course of blood pressure after administration of a striated muscle trypsin hydrolyzate (50 mg peptide / kg). FIG. 5 shows striated muscle trypsin digest (1
5 shows the change over time in blood pressure after administration of (00 mg peptide / kg). FIG. 6 shows gonad pepsin degradation product (50 m
(g peptide / kg) administration shows the time-dependent change of the blood pressure. FIG. 7 shows gonad pepsin degradation product (100 mg).
2 shows the time course of blood pressure after administration of (peptide / kg). As can be seen from FIGS. 4 to 7, it was observed that both the peptide mixture obtained from the striated muscle and the gonad lower the blood pressure of spontaneously hypertensive rats (SHR). For peptide mixtures obtained from gonads, 10
It is considered that the drop is strong in the order of 0 and 50 mg peptide / kg and the concentration is dependent. On the other hand, for a peptide mixture obtained from striated muscle, 100 mg pe
50mg peptide compared to ptide / kg group
/ Kg, the blood pressure drop was remarkable, and the average blood pressure decreased by 35 mmHg when compared with 6 hours after administration, which showed the lowest blood pressure, and before administration.

<Purification of Physiologically Active Peptide and Confirmation of Its Antihypertensive Effect> An ACE inhibitory active peptide was isolated by chromatography from enzymatic digestion of striated muscle oyster muscle. The amino acid composition and sequence of the obtained ACE inhibitory activity peptide were examined. ACE obtained from enzyme-treated striated muscle
Inhibitory peptides include Asp (aspartic acid), Le
u (leucine), Thr (threonine), and Tyr (tyrosine), and the ratio is 2:
1: 1: 1 and the sequence was Asp-Leu-Thr-
Asp-Tyr. IC of this new peptide
₅₀ was 0.143 μmol / ml. Table 1 shows a comparison between this peptide and other known ACE inhibitory peptides (from sardine muscle and from clam soft body).

[Table 1] In addition, the searched peptide was chemically synthesized, and an oral administration experiment to spontaneously hypertensive rats (SHR) was performed. FIG. 8 shows the result. FIG. 8 shows that striated muscle-derived peptide (synthetic compound) was administered to spontaneously hypertensive rats (8 mg pept).
It is a figure which shows the time-dependent change of the blood pressure after (ide / kg).
As shown in this figure, the blood pressure showed the lowest value 6 hours after administration, and an average decrease of 15 mmHg was observed as compared with that before administration. As a result, it was confirmed that the peptide having the ACE inhibitory activity in the in vitro system has a lowering effect also in the in vivo system.

<Dipeptide> The above-mentioned pentapeptide Asp-Leu-Thr-Asp-Tyr derived from Japanese oyster is
ACE inhibitory activity in vitro and blood pressure lowering effect in vivo have been confirmed, but since this pentapeptide is a long-chain peptide, it is highly likely that it actually works in the form of di- and tripeptides . Therefore, the in vivo degradation mode and action mechanism of the pentapeptide were examined. The oyster-derived pentapeptide Asp-Leu-Thr
-Four dipeptides that may be derived from Asp-Tyr Asp-Leu (DL), Leu-Thr (L
T), Thr-Asp (TD) and Asp-Tyr
(DY) was synthesized, and the ACE inhibitory activity in vitro was measured. Each peptide was adjusted to a concentration of 1 mM. The measurement of the ACE inhibitory activity was carried out according to the method of Yamamoto et al., Which was modified from Lieberman's assay, and was expressed as the inhibitory activity against the substrate Hip-His-Leu and rabbit lung ACE. FIG. 9 shows the results.
It was shown to. DY has the strongest inhibitory activity, followed by TD and D
L, then LT.

<Dipeptide digestive enzyme resistance and A after digestion
CE inhibitory activity> For the above four dipeptides, digestion resistance to digestive enzymes in vivo and A by digestive enzyme treatment
Changes in CE inhibitory activity were examined. The digestion test was performed according to the following procedure. In 16 μmol of each peptide, 2.0 ml of a pepsin solution (0.1 mg / ml 0.1N HC
l) was added and incubated for 3 hours at 37 ° C. 0.
After neutralization by adding 1 ml of 2N NaOH, 1 ml of a pancretian solution (4.47 mg / ml-0.2 M) was neutralized.
MOPS, pH 8.0) to the reaction mixture,
The reaction was stopped by incubating at 10 ° C. and boiling for 10 minutes. This was subjected to ultrafiltration (2000 rpm, 10 min) using Ultrafree, and the resulting filtrate was measured for ACE inhibitory activity and the amount of amino acids generated by peptide degradation. Amino acid amount is TNBS
(Trinitrobenzene-sulfone) method.
The TNBS method was performed according to the following procedure. Dried sample (3 ~
100 µl of reagent A
(0.25 M borate buffer: 2.5 M NaOH <2
0: 1>), followed by 5 ml of TNBS reagent (35 mg of recrystallized TNBS is dissolved in 1 ml of 0.25 M borate buffer just before use). 90 minutes after 10 minutes
0 μl of reagent B (62.5 mg K ₂ SO ₃ just before use)
Is dissolved in 0.2 M KH ₂ PO ₄ to make 100 ml. ) Was added and the absorbance at 416 nm was measured.
The ACE inhibitory activity of each peptide after the enzyme treatment is shown in FIG. As a result, DY had the strongest inhibitory activity, and the other peptides had weaker activities. In the change in activity after enzyme treatment,
DY and LT showed little change in activity, whereas TD and DL showed a large decrease in activity due to enzyme treatment. FIG. 11 shows the results of the resistance to degradation of the peptide by the enzyme treatment. As a result, of the four peptides, DY had the highest degradation resistance and LT was weak. Judging from the degradation resistance of each peptide and the change in ACE inhibitory activity due to the enzyme treatment, DY showed the strongest enzyme resistance, and as a result, almost no loss of ACE inhibitory activity was observed. TD and DL have weak enzyme resistance and ACE
The loss of inhibitory activity was also significant. Although LT has remarkably low enzyme resistance, ACE inhibitory activity was not significantly changed. This is also considered to mean that LT is almost degraded and contains ACE inhibitory activity, although it is weak in degradation products (amino acids).
Details are unknown.

<Pentapeptide Digestion Test> Based on the above results, a Japanese oyster pentapeptide was treated in the same manner to clarify the stability and degradation products of the peptide. First, after examining the separation conditions in HPLC (high performance liquid chromatography), the pentapeptide degradation product was subjected to HPLC. HPLC conditions are as follows. Column: ODS80TM (0.46 × 25 cm) Eluent: (A); 0.05% TFA + 4% CH ₃ CN (B); 0.05% TFA + 25% CH ₃ CN Linear gradient (A) to (B), 30 min Flow rate: 1.0 ml / min Column temperature: 40 ° C. As a result, according to the chromatograph, the pentapeptide is easily decomposed by the two-step digestion enzyme treatment used in this study, and has high stability in decomposed organisms. DY having a strong inhibitory activity was confirmed.

<Administration test using spontaneously hypertensive rats (SHR)> Based on the results obtained in vitro, a degradation product of DY and pentapeptide, which can be expected to have a hypotensive effect in vivo, was orally administered to spontaneously hypertensive rats (SHR). It was subjected to an administration test to confirm its antihypertensive effect. The administration test consisted of 6 animals per group, and blood pressure was measured immediately after administration and at 3, 6, 9, and 24 hours after administration by the Tail-Cuff method using a non-invasive tail arterial blood pressure measurement device. The results are shown in FIGS. In an in vivo blood pressure lowering test using spontaneously hypertensive rats (SHR), Asp-Tyr (DY) and pentapeptide hydrolyzate showed a blood pressure lowering of about 20 mmHg. This indicates that the oyster pentapeptide Asp-Leu-T
It was presumed that hr-Asp-Tyr was decomposed in the intestinal tract by digestive enzymes, converted to a peptide having a smaller molecular weight, and acted. In this case, Asp- which has strong degradation resistance as a dipeptide and has the strongest blood pressure lowering action
It is presumed that Tyr (DY) exerts a blood pressure lowering action as a final acting molecule.

<ACE Inhibition Mode of Asp-Tyr (DY)> The ACE inhibition mode of DY, which exhibited activity in vitro and in vivo, was clarified. The method determines the relationship between the substrate concentration and the reaction rate when the inhibitory peptide DY is added to the reaction mixture, and uses the data obtained to determine
The mode of inhibition was determined by aver-Burk blot.
As a result, it was revealed that the mode of DY inhibition was non-antagonistic. Specifically, by binding of DY to the ACE molecule, the three-dimensional structure of the andeotensin I binding site in the ACE molecule (conformation)
Is changed, and inhibition by angiotensin I binding is considered to be an inhibition.

[0013]

EFFECT OF THE INVENTION By mixing a peptide of the present invention derived from a Japanese oyster having an antihypertensive effect into foods and drinks,
Foods and drinks having a blood pressure lowering effect can be obtained. ACE inhibitors derived from natural products such as the present invention can be expected to have few side effects.

[Brief description of the drawings]

FIG. 1 is a diagram showing screening of a oyster gonad extract using an enzyme.

FIG. 2 is a graph showing the ACE inhibitory activity of each tissue of a oyster treated with pepsin.

FIG. 3 is a graph showing the ACE inhibitory activity of each tissue of a Japanese oyster treated with trypsin.

FIG. 4: Decomposition product of striated muscle trypsin (50 mg pept)
FIG. 2 is a graph showing the time course of blood pressure of spontaneously hypertensive rats (SHR) after oral administration of (ide / kg).

FIG. 5: Striated muscle trypsin digest (100 mg pep)
FIG. 2 is a graph showing the time-dependent changes in blood pressure of spontaneously hypertensive rats after oral administration (ide / kg).

FIG. 6 shows gonad pepsin degradation product (50 mg pepti)
de / kg) after oral administration, spontaneously hypertensive rats (S
It is a figure which shows the time-dependent change of the blood pressure of (HR).

FIG. 7 shows gonad pepsin degradation product (100 mg pept)
(ide / kg) to spontaneously hypertensive rats (SHR) after administration.

FIG. 8 shows administration of striated muscle-derived peptide (synthetic compound) to spontaneously hypertensive rats (SHR) (8 mg peptide /
It is a figure which shows the time-dependent change of the blood pressure after (kg).

FIG. 9 is a graph showing ACE inhibitory activities of four synthetic dipeptides derived from a pentapeptide.

FIG. 10 shows the ACE inhibitory activity of dipeptides after enzyme treatment.

FIG. 11 is a diagram showing digestive enzyme resistance of dipeptides.

FIG. 12 is a graph showing a test of administration of Asp-Tyr (DY) and a pentapeptide degradation product to spontaneously hypertensive rats (SHR).

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-10-150923 (JP, A) JP-A-8-225593 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07K 5/06-5/072 C07K 7/06 A61K 38/05-38/08 CA (STN) BIOSIS (DIALOG) REGISTRY (STN)

Claims (1)

(57) [Claims] (1) the amino acid sequence is Asp-Leu-Thr-
A peptide having an antihypertensive effect, which is Asp-Tyr or Asp-Tyr.