JPH0778077B2 - New peptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel peptide, more specifically to a peptide having an excellent antihypertensive action, which is useful as a pharmaceutical product, a process for producing the same, and its use.

[Conventional technology and its problems]

Conventionally, active ingredients have been extracted and collected from the tissues of fish, and some of them have already been put to practical use as pharmaceuticals and the like. Moreover, there is a possibility that undiscovered physiologically active substances may exist, and the discovery thereof is desired.

[Means for Solving the Problems]

In such a situation, the present inventor has conducted a long-term study on components in fish tissues, and found that a particular extracted component of fish tissues has a strong antihypertensive effect. Then, as a result of intensive research to clarify the component, it was confirmed that this was a peptide represented by the formula (I) described below, and the present invention was completed.

That is, the present invention provides a peptide represented by the following formula (I), His-Gln-Ala-Ala-Gly-Trp (I), a method for producing the peptide, and an antihypertensive agent containing the peptide.

The peptide (I) of the present invention can be isolated and purified from fish tissue, for example, as follows.

The water extract component of fish is treated with a proteolytic enzyme and then purified to produce the peptide (I) of the present invention.

Sardines, particularly sardines, are preferable as the fish. In addition, water-exposed waste liquid obtained in the production process of surimi can also be used. As the protease, acidic protease, serine protease and the like can be used, but pepsin is particularly preferable.

As the method for purifying the peptide (I) of the present invention, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography and the like are used alone or in combination in any order.

An example of the extraction method using sardine as a raw material is as follows.

After adding distilled water to the ordinary meat of sardine, stirring and extracting,
Centrifuge and add pepsin to the resulting supernatant for digestion.
This pepsin digest was treated with octadecylsilanated (ODS) carrier for reversed phase chromatography, and SP-
Peptide (I) can be obtained by purification through a column of Sephadex C-25 and Sephadex G-10 and further separation by high performance liquid chromatography (HPLC).

The peptide (I) of the present invention can also be produced by binding the constituent amino acids according to a conventional peptide synthesis method.

As the peptide synthesis method, either a liquid phase method or a solid phase method may be used, and a commercially available peptide synthesizer may be used. Peptide (I) can be purified and isolated by reverse phase column chromatography which is usually used for peptide separation.

The peptides represented by the formula (I) show excellent ACE inhibitory activity regardless of their origin and are useful as antihypertensive agents.

As its usage, intravenous administration or oral administration is preferable.

Usable dosage forms include injections or capsules, tablets, powders, oral administration agents such as granules.

Although the optimal dose of the peptide (I) of the present invention varies considerably depending on the degree of symptoms of patients, administration method and the like, the therapeutic daily dose for an adult is 1.0 to 1000 mg.

The peptide (I) of the present invention can be made into a pharmaceutical preparation by a conventional method together with any conventional pharmaceutical carrier, base or excipient. It is preferable to use as fat-soluble capsules, tablets, powders or granules for oral administration and as water-soluble injections or freeze-dried powders as injections. Although a conventional substance can be used as an excipient, human serum albumin, sucrose, gelatin, starch, polyethylene glycol or the like is preferably used.

〔Example〕

Next, the present invention will be described more specifically with reference to Examples.
The present invention is not limited to these.

In this Example, the blood pressure lowering effect was measured by Cushma.
n &Cheung's method [Biochem. Pharmacol., 20, 1637 (1
971)] was used to measure angiotensin I converting enzyme (ACE) inhibitory activity.

ACE1-10mU, Substrate Hip-His-Leu (Peptide Institute)
2.5 mM, potassium phosphate buffer (pH 8.3) 100 mM, salt 300
A predetermined amount of the sample was added to a solution containing mM, the mixture (0.25 ml) was incubated at 37 ° C. for 30 minutes, and then 0.25 ml of 1N hydrochloric acid was added to stop the enzymatic reaction. Ethyl acetate 1.5m
l was added and stirred vigorously for 10 seconds to extract the produced hippuric acid. The solvent was distilled off from the ethyl acetate layer (1 ml), and the residue was distilled water (1 ml).
It was dissolved in ml and the absorbance at 228 nm was measured (ODs). Before the incubation, the one to which 1N hydrochloric acid had been added in advance was used as a blank (ODsbl), and the difference in absorbance (ODs-ODsbl) was determined. In addition, a sample to which no sample was added was used as a control group (ODc), and (ODc-ODcbl) was similarly obtained. The ACE inhibitory activity was calculated by the following formula.

Example 1 (1) 200 g of ordinary sardine meat was sliced and homogenized by adding 3 volumes of distilled water and then centrifuged at 4,000 × g for 20 minutes to collect a supernatant. The supernatant is passed through an ultrafiltration membrane (molecular weight 5,000
Fractionation), adjust the pH to 3.0 with HCl, add 1/100 of the total protein (bovine serum albumin equivalent by protein assay) of the concentrated solution, and add at 25 ° C for 24 hours. Digested for hours. After heating at 100 ° C. for 10 minutes to inactivate pepsin, centrifugation was performed at 15,000 × g for 20 minutes, and the obtained supernatant was freeze-dried to obtain 9.6 g of a pepsin digest. The ACE inhibitory activity IC ₅₀ of this product was 184 μg / ml.

(2) The lyophilized powder of (1) was redissolved in distilled water and adsorbed on a column (φ2.4 × 23 cm) packed with pretreatment ODS,
After washing with distilled water 1, it was eluted with 800 ml of 15% acetonitrile. The eluate was freeze-dried to obtain 2.1 g of Fr.I. The ACE inhibitory activity IC ₅₀ of this product was 176 μg / ml.

(3) Fr.I was dissolved in 0.2M acetic acid and SP-Sephadex C was added.
Pass through a column (5.0 x 25 cm) packed with -25, and 0.7 ml / min
Gradient elution of pH = 3.1 to 5.0 was performed with 0.2 M pyridine-acetic acid buffer at a flow rate of. The elution pattern is as shown in FIG. The fraction with the highest ACE inhibitory activity per weight was collected and designated as Fr.II. This product has an ACE inhibitory activity IC ₅₀ of 94 μg /
It was ml.

(4) Fr.II was passed through a column (1.6 × 95 cm) packed with Sephadex G-25 and eluted with 0.2 M acetic acid at a flow rate of 6 ml / h. The elution pattern is as shown in FIG. Fractions having ACE inhibitory activity were collected to obtain Fr.III. ACE of this thing
The inhibitory activity IC ₅₀ was 52 μg / ml.

(5) Fer.III with Develosil ODS-10 column (2.0 x 25 cm)
HPLC (eluent: 0.05% TFA solution containing 10 to 20% acetonitrile). The elution pattern is the third
As shown in the figure. The peak portion having ACE inhibitory activity was collected and designated as Fr.IV. This product has an ACE inhibitory activity IC ₅₀ of 41 μg /
It was ml.

(6) Use Fr.IV with Asahi Pack GS-320 column (0.76 × 50c
m) was used for HPLC (eluent: 50 mM ammonium acetate-acetic acid buffer pH = 6.7). The elution pattern is as shown in FIG. Fr.V was obtained by collecting peaks showing ACE inhibitory activity. This was freeze-dried to give 0.4 m of the hexapeptide of the present invention.
got g.

Amino acid composition: Hydrolyzed with 6N hydrochloric acid or 4M methanesulfonic acid at 110 ° C for 24 hours, and measured with a Hitachi L-8500 amino acid analyzer. The results are shown in Table 1.

Amino acid sequence: The result determined using a gas phase protein sequencer model 470A (Applied Biosystems) is as shown in the following formula (I).

His-Gln-Ala-Ala-Gly-Trp (I) Example 2 Using a fully automatic peptide synthesizer model 430A (Applied Biosystems), the following formula (I) His-Gln-Ala-Ala-Gly-Trp ( The peptide represented by I) was synthesized. The peptide (I) of the present invention was purified by reverse phase HPLC using an ODS column after elimination from the resin carrier and removal of the protective group with hydrogen fluoride.

As in Example 1, the result of measuring the amino acid composition was in good agreement with the value expected from the sequence of peptide (I).

Example 3 The peptide (I) obtained in Example 2 was dissolved in physiological saline, and 10 6-week-old male and female ICR mice and 10 6-week-old male and female Wistar rats, respectively, were used. Various concentrations of peptide (I) were intravenously administered to the group of experimental animals, and the LD ₅₀ value was measured after 2 weeks of observation.

As a result, the LD ₅₀ value of peptide (I) was higher than 100 mg / kg in both mouse and rat.

As is clear from the above examples, the peptide (I) of the present invention is a safe and extremely useful antihypertensive substance that exhibits excellent ACE inhibitory activity and has low toxicity.

Specific utility of the peptide (I) of the present invention described above will be described below with reference to Examples for medical use.

Example 4 Water-soluble injection 100 mg of peptide (I) obtained in Example 2 was dissolved in 100 ml of 0.01 M phosphate buffer (pH 7.0) containing 0.14 M sodium chloride prepared with distilled water for injection. This solution was aseptically filtered using a membrane filter, and 1 ml each of the filtrate was filled in a glass container and sealed to give a water-soluble injection.

Example 5 Tablets Peptide (I) obtained in Example 1 50 g Corn starch 20 g Lactose 50 g Microcrystalline cellulose 23 g Hydroxypropyl cellulose 5 g Light anhydrous silicic acid 1 g Magnesium stearate 1 g From the above ingredients, 50 mg of the peptide (I) of the present invention To obtain 1000 tablets.

That is, the above components were mixed by a conventional method, and then granulated and tabletted into 1000 tablets so that each of them would be 150 mg. The tablets were further coated with hydroxypropylmethylcellulose, talc, titanium dioxide and sorbitan fatty acid ester according to a conventional method to obtain 1000 coated tablets.

Example 6 Capsule Preparation Peptide (I) obtained in Example 2 50 mg Magnesium stearate 7 mg Lactose 243 mg The above ingredients were mixed by a conventional method and filled in No. 1 gelatin capsule to obtain a capsule preparation.

[Brief description of drawings]

Figure 1 shows the sardine ordinary meat water extract after pepsin treatment,
Fr.I obtained by ODS treatment was SP-Sephadex C-25.
Fig. 2 shows the elution pattern when subjected to ion exchange chromatography, and Fig. 2 shows the elution pattern when subjected to Sephadex G-25 gel filtration chromatography with Fr.II.
The figure shows HPL using Fr.III Develosil ODS-10 column.
FIG. 4 is a C pattern, FIG. 4 is an HPLC pattern when using an Asahi Pack GS-320 column of Fr. IV, and FIG. 5 is a drawing showing an ultraviolet absorption spectrum of the peptide (I) of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display area C07K 1/16 // C07K 123: 00

Claims (6)

[Claims] 1. A peptide represented by the following formula (I): His-Gln-Ala-Ala-Gly-Trp (I). 2. The peptide according to claim 1, which is obtained by purifying an extract from fish tissue. 3. The peptide according to claim 1, which is obtained by binding constituent amino acids by a peptide synthesis method. 4. The method for purifying a peptide according to claim 1, wherein at least one of the following steps is performed in an arbitrary order for the liquid extracted from the tissue of fish. Ion exchange chromatography Gel filtration chromatography Reversed phase chromatography 5. The method for producing a peptide according to claim 1, wherein the constituent amino acids are peptide-bonded by a peptide synthesis method of a liquid phase method or a solid phase method. 6. A hypotensive agent comprising the peptide according to any one of claims 1 to 3 as an active ingredient.