JPH07119234B2 - Tripeptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to a novel tripeptide, more specifically to a tripeptide having an excellent antihypertensive effect, which is useful as a pharmaceutical product, a method for producing the same, and its use. .

[Problems to be Solved by Conventional Techniques and Inventions]

Conventionally, it is known that seafood contains components having a therapeutic effect or a preventive effect on hypertension. For example, also for Antarctic krill, the electrolyte extract has an effect of preventing hypertension (Japanese Patent Publication No. 63-47690).
(JP-A No. 137952), or foods containing the protein have been reported to prevent hypertension (JP-A-1-137952).

However, the substance of the active ingredient contained in Antarctic krill has not been clarified, and its elucidation and provision of a new antihypertensive agent have been desired.

[Means for Solving the Problems]

In such a situation, the present inventor has conducted a long-term study on the components in the tissue of Antarctic krill, and found that a specific extract component of Antarctic krill tissue has a strong antihypertensive effect. Then, as a result of further diligent research to elucidate the component, it was confirmed that this was a tripeptide represented by the formula (I) described below, and the present invention was completed.

That is, the present invention provides a tripeptide represented by the following formula (I) Leu-Lys-Tyr (I), a method for producing the same, and a hypotensive agent containing the same.

The tripeptide (I) of the present invention can be obtained, for example, by purifying an extract from the tissue of Antarctic krill.

That is, the tripeptide (I) of the present invention can be obtained by treating the fraction obtained from Antarctic krill with a proteolytic enzyme and then purifying. Examples of the proteolytic enzyme used here include acid protease, serine protease and the like, but pepsin and trypsin are particularly preferable, and these proteolytic enzymes are used alone or in combination. As the method for purifying the tripeptide (I) of the present invention, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, etc. may be used alone or in combination in any order.

An example of the extraction and purification method in the case of using the ground meat of Antarctic krill as a raw material is as follows.

After heating the peeled Antarctic krill, it is exposed to water to remove water-soluble components, and then defatted with acetone and dried. Distilled water is added to this dry powder to suspend it, treated with pepsin, and then centrifuged to collect the supernatant. The obtained supernatant is treated with trypsin, purified through a column of ODS-modified silica gel and SP-Sephadex C-25, and then separated by high performance liquid chromatography (HPLC) to obtain tripeptide (I).

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

In the present invention, 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. Tripeptide (I) can be purified and isolated by reverse phase column chromatography which is usually used for peptide separation.

The thus obtained tripeptides 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.

The dose of the tripeptide (I) of the present invention varies considerably depending on the degree of symptoms of the patient, the administration method, etc.
The usual daily therapeutic dose for adults is 1.0 to 1000 mg.

The tripeptide (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〕

The present invention will be described more specifically with reference to the following examples.
The invention is in no way limited by these examples.

In this Example, the blood pressure lowering effect was determined by Cushmann &
Cheung's method [Biochem. Pharmacol., 20, 1637 (197
1)] was used to measure angiotensin I converting enzyme (ACE) inhibitory activity.

That is, ACE1 to 10 mU, substrate Hip-His-Leu (manufactured by Peptide Institute) 2.5 mM, potassium phosphate buffer (pH 8.3) 100 m
A predetermined amount of the sample was added to a solution containing M and 300 mM of sodium chloride, 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. 1.5 ml of ethyl acetate was added and vigorously stirred for 10 seconds to extract the hippuric acid produced. The solvent was distilled off from 1 ml of the ethyl acetate layer, the residue was dissolved in 1 ml of distilled water, and the absorbance at 228 nm was measured (OD
s). Before incubating, 1N hydrochloric acid was added in advance as a blank (ODsbl) and the difference in absorbance (ODs-OD
sbl). 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 (i) 100 g of frozen frozen Antarctic krill at 80 ° C.
After heating for 10 minutes, 34 g of dehydrated meat was obtained. This was degreased with acetone, filtered, and vacuum dried at 60 ° C. to obtain 9.9 g of Antarctic krill protein powder.

To the Antarctic krill protein powder, 20 volumes of water was added, pH was adjusted to 3.0 with HCl, 1/100 volume of pepsin was added, and digested at 45 ° C. for 24 hours. After heating at 100 ° C. for 10 minutes to inactivate pepsin, centrifugation was performed at 15000 × g for 20 minutes, and the collected supernatant was freeze-dried to obtain 4.6 g of a pepsin digest. A 20-fold amount of 1 M ammonium bicarbonate solution was added to the digested product of pepsin to dissolve it, and 1/100 amount of trypsin was added thereto, followed by digestion at 37 ° C. for 15 hours. After heating at 100 ° C for 10 minutes to inactivate trypsin, lyophilize the digested product.
Obtained 4.5 g. Its ACE inhibitory activity IC ₅₀ was 106 μM.

(Ii) Redissolve the freeze-dried powder of (i) in distilled water,
It was adsorbed on an ODS 24S column (φ2.4 × 23 cm), washed with 800 ml of distilled water, and then eluted with 900 ml of 15% acetonitrile. Lyophilize the eluate of the fraction having ACE inhibitory activity,
Fraction I 1.9 g was obtained. ACE inhibitory activity IC of this product
₅₀ was 95 μM.

(Iii) Fraction I (100 mg) was dissolved in 0.2 M acetic acid, and SP-
Column packed with Sephadex C-25 (φ1.5 × 16c
m) and 0.02 M ammonium acetate-acetic acid buffer (pH 3.0) to 0.3 M ammonium acetate-acetic acid buffer (pH 6.
(0) was used for gradient elution, and the fraction with high ACE inhibitory activity was freeze-dried to obtain 17 mg of Fraction II. The elution pattern is as shown in FIG. Its ACE inhibitory activity IC ₅₀ was 68 μM.

(Iv) Fraction II 6 mg was subjected to HPLC using Super Rose 12 column (φ1.0 × 30 cm), eluted with 0.2 M acetic acid, and the fraction with high ACE inhibitory activity was lyophilized,
Fraction III 0.7 mg was obtained. The elution pattern is as shown in FIG. This product has an ACE inhibitory activity IC ₅₀ of 55.
μM.

(V) Fraction III 0.7 mg was added to ODS-5 column (φ0.46
X25 cm) and subjected to gradient elution using 0.05% trifluoroacetic acid to 20% acetonitrile-containing 0.05% trifluoroacetic acid to collect a fraction with high ACE inhibitory activity,
Fraction IV 0.03 mg was obtained. The elution pattern is as shown in FIG.

Further, 0.03 mg of Fraction IV was subjected to HPLC again under the same conditions to obtain a single peak, Fraction V. The elution pattern is as shown in FIG. Fraction V was freeze-dried to obtain 0.02 mg of the tripeptide of the present invention. The ACE inhibitory activity IC ₅₀ of the obtained tripeptide of the present invention was 4.5 μM.

Amino acid composition: The obtained tripeptide of the present invention was hydrolyzed with 6N hydrochloric acid at 110 ° C. for 22 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).

Leu-Lys-Tyr (I) Example 2 Using a fully automatic peptide synthesizer model 430A (Applied Biosystems), a tripeptide represented by the following formula (I) Leu-Lys-Tyr (I) was synthesized. The tripeptide (I) of the present invention was removed from the resin carrier by hydrogen fluoride and the protecting group was removed, and then reversed phase HPLC using an ODS column was performed.
Was purified.

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

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

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

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

Regarding the tripeptide (I) of the present invention described above,
The specific utility will be described below with reference to examples for medical use.

Example 4 Water-soluble injection 100 mg of the tripeptide (I) obtained in Example 2 was dissolved in 100 ml of 0.014M phosphate buffer (pH 7.0) containing 0.14M sodium chloride prepared with distilled water for injection. . This solution was aseptically filtered using a membrane filter, and the filtrate was filled in a glass container in an amount of 1 ml and sealed to give a water-soluble injection.

Example 5 Tablets Tripeptide (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, the tripeptide (I) of the present invention 1000 tablets containing 50 mg were obtained.

That is, the above ingredients were mixed by a conventional method, granulated, and then 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 by a conventional method to obtain 1000 coated tablets.

Example 6 Capsule Preparation Tripeptide (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 into No. 1 gelatin capsule to obtain a capsule preparation.

〔The invention's effect〕

As mentioned above, the tripeptide (I) of the present invention has excellent AC
It has E inhibitory activity and is useful as an antihypertensive agent.

[Brief description of drawings]

Fig. 1 shows LOP of digested enzyme products of Antarctic krill protein.
The elution pattern of Fraction I fractionated with ODS 24S column was purified using SP-Sephadex C-25 column. Fig. 2 shows the elution pattern of Fraction II purified with HPLC using Superrose 12 column. FIG. 3 shows the elution pattern when fraction III was subjected to HPLC using develocyl ODS-5, and FIG. 4 is the elution pattern when fraction IV was re-eluted on the same column.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display area C07K 1/18 1/20 1/34

Claims (6)

[Claims] 1. A tripeptide represented by the following formula (I): Leu-Lys-Tyr (I). 2. The tripeptide according to claim 1, which is obtained by purifying an extract from the tissue of Antarctic krill. 3. The tripeptide according to claim 1, which is obtained by binding constituent amino acids by a peptide synthesis method. 4. The method for purifying a tripeptide according to claim 1, wherein at least one of the following steps is performed in a liquid extracted from the tissue of Antarctic krill in any order. Ion exchange chromatography Gel filtration chromatography Reversed phase chromatography 5. The method for producing a tripeptide according to claim 1, wherein the constituent amino acids are bound to the peptide by a peptide synthesis method such as a liquid phase method or a solid phase method. 6. A blood pressure lowering agent comprising the tripeptide according to any one of claims 1 to 3 as an active ingredient.