JPH01240170A - Composition for peroral administration - Google Patents

Abstract

PURPOSE:To obtain the subject composition for peroral administration useful as a health food, etc., for the prevention of hypertension, etc., by compounding a peptide capable of inhibiting angiotensinase. CONSTITUTION:Swine plasma is adjusted to pH 1-2 and hydrolyzed with a proteinase to obtain a peptide capable of inhibiting angiotensinase. The peptide is used singly or in combination with egg yolk to obtain the objective composi tion having proper form and shape. Usually a male adult is administered with 1-500mg of said peptide and 5mg-2g of the egg yolk daily per 1kg of the body weight.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an oral food useful as a health food for preventing hypertension.

[Conventional technology]

Today, hypertension is one of the diseases with a high mortality rate in Japan, and its treatment or prevention has become an urgent and important issue.

Hypertension includes secondary hypertension and essential hypertension, and the onset and pathology of the former, such as renal hypertension or endocrine hypertension, and the latter, essential hypertension, are both related to blood pressure. It is well known that the intermediate active peptide producing systems, especially the renin-angiotensin system, are closely related. This renin-angiotensin system contains angiotensin converting enzyme (Ang), which is involved in blood pressure 1) 1J.
iotensin Converting Enzym
e, hereinafter referred to as ACE) is present, and this enzyme produces an active peptide (angiotensin ■) that has a smooth muscle contraction effect on blood vessel walls.
A strong increase in blood pressure is brought about.

Therefore, it is thought that if this enzyme activity is inhibited, it will be possible to control the increase in blood pressure (lower blood pressure), and from this point of view, the search for inhibitors of various natural and synthetic substances is underway, and has already been carried out. Regarding synthetic compounds, certain types of proline derivative compounds have been recognized for their effectiveness and are put into practical use as antihypertensive agents.

On the other hand, it has been reported that, as an ACE inhibitor derived from a natural product, an ACE inhibitory peptide exists in a trypsin-digested product of casein (Japanese Unexamined Patent Publication No. 58-109425).

Since these naturally derived ACE inhibitors are obtained from foods or food raw materials, they are expected to be low-toxic and highly safe antihypertensive agents, and their practical use as antihypertensive agents is being considered. There is.

However, sufficient results have not yet been obtained.

[Problem to be solved by the invention]

In view of the difficulty in fundamentally curing hypertension, especially essential hypertension, the present inventors have been working on daily and continuous control of blood pressure (for example, the intake of health foods that have a blood pressure lowering effect). Focusing on the importance of preventing the onset of hypertension (e.g., hypertensive disorders, etc.) and alleviating hypertensive tendencies, we are searching for antihypertensive substances that can be used for such purposes, have low toxicity, and are effective when administered (ingested) orally. However, considering the excellent properties of the above-mentioned ACE inhibitory peptide derived from pig plasma, especially its low toxicity, we investigated the possibility of using it for the above purpose. A hypotensive effect was observed upon administration, and furthermore, it was found that the antihypertensive effect was enhanced by administering the peptide in combination with egg yolk. We have discovered that this, combined with its moderate hypotensive effect, low toxicity, and high safety, makes it possible to provide an oral food product that is useful as a health food for preventing hypertension and alleviating hypertension tendencies. The invention was completed.

[Structure of the invention]

That is, the present invention is an oral feeding composition characterized by containing a peptide that inhibits angiotensin converting enzyme obtained by decomposing pig plasma with a protease, and further comprising an egg yolk in the above composition. The present invention relates to an oral food product characterized by containing.

Obtained by proteolyzing and enzymatically decomposing pig plasma.
A peptide that inhibits angiotensin converting enzyme is prepared, for example, as follows.

Pig plasma is adjusted to P H1-P H2 and hydrolyzed by the action of proteolytic enzymes. Add excess methanol to this decomposition solution and remove the formed precipitate. After concentrating the supernatant under reduced pressure, a purified peptide is obtained using an ion exchange resin column, gel filtration, or the like.

The proteases used include pepsin, trypsin, chymotrypsin, papain, and promelain, as well as bacterial proteases (subtilisin, thermolysin, nagase, etc.). These enzymes can be used alone or in combination.

The peptide thus obtained has a molecular weight of 500 to
5,000 and has ACE inhibitory activity.

As described above, the peptides derived from pig plasma of the present invention are
This product is provided for oral consumption as it is, or more preferably as a composition in an appropriate shape and form together with a suitable non-toxic carrier for oral administration (ingestation).

Examples of compositions include ACE-inhibiting peptides together with pharmaceutically acceptable carriers (excipients, lubricants, binders, colorants, flavoring agents, excipients, etc.) in pharmaceutical formulations for oral administration. Examples include tablets (tJ! coated tablets, effervescent tablets, film-coated tablets, chewable tablets, etc.), capsules, troches, powders, fine granules, granules, etc.

It may also be in the form of solid or liquid foods or luxury goods, such as confectionery, powdered tea, ice cream, yogurt, alcoholic beverages, sports drinks, and the like.

The content of ACE inhibitory peptide in oral food is
It can be selected as appropriate depending on the dosage form, but generally 1 to 10
It is in the range of 0% by weight.

As shown in test examples later, the oral food of the present invention having the above-mentioned structure exhibits a good antihypertensive effect even when administered orally, and has extremely low toxicity. It can be orally administered (ingested) continuously for the purpose of alleviating or regulating blood pressure, and is expected to be effective when used as a health food for preventing hypertension.

When the oral food of the present invention is used for such purposes, the intake amount is 1.0 mg to 50 mg per day for adult males.
A range of 0 mg/Kg body weight) is suitable.

The present inventors further discovered that the use of the active peptide and egg yolk in combination resulted in improvement over time.

That is, the yolk of a commercially available chicken, quail, or ostrich egg may be used. Also, egg yolk can be used in liquid or powdered form.

In this case, the content ratio of the above-mentioned peptide and egg yolk in the oral feeding composition of the present invention is 1:0. A range of 3 to 1:1O is preferred.

Examples of compositions according to the invention are the same as those already mentioned above, i.e. the active ingredient together with a pharmaceutically acceptable carrier in the form of a tablet preparation for oral administration (e.g. tablets, etc.);
Moreover, it may be in the form of a food or a luxury item.

The contents of the active peptide and egg yolk in the oral composition of the present invention can be appropriately selected depending on the dosage form, but the total content of -a is in the range of 5 to 100% by weight.

Further, in this case, the intake amount is preferably in the range of 1 mg to 500 mg/Kg body weight per day for an adult male in terms of weight of the peptide, and the range of egg yolk is 5 mg to 2 g/Kg body weight per day.

Below, examples of the production of the ACE-inhibiting peptides derived from pig plasma, which are the active ingredients of the oral food of the present invention, and animal experiments showing that oral administration of these peptides alone and in combination with egg yolk (blood pressure The results of the descent test) are listed below.

In addition, the activity (!D,.) of the ACE inhibitory peptide was measured by the following method.

[Measurement of ACE inhibitory activity of ACE inhibitory peptide] i) Preparation of angiotensin converting enzyme solution (ACEI) 5 g of rabbit lang acetone powder (manufactured by Sigma)
of 50m2. lM borate buffer (PH 8.
3), centrifuged at 40,000xg for 40 minutes, and the supernatant was further dissolved in the above buffer for 50 minutes.
The solution was diluted twice to obtain an angiotensin converting enzyme solution.

ii) Measurement of activity: Place the sample in a test tube at 0.00%. 0 3 ml of 250 μ2 of hybrillium, one histidyl, and one leucine as a substrate [Andrich Chemical Co., Ltd. (Aldrich
CheIl. Co. ), final concentration 5mM.

Contains 300mM NaCl. ] at 37°C.
After incubating for 0 minutes, the enzyme solution was heated to 0. Add 1 ml,
The reaction was carried out at 37°C for 30 minutes. Then, 1N hydrochloric acid 0.
After stopping the reaction by adding 25 ml, 1.
5 ml of ethyl acetate was added and stirred vigorously for 15 seconds. Then, centrifuge at 3.50 rpm for 15 minutes.
1 ml of ethyl acetate was collected. The ethyl acetate layer was heated at 120°C for 30 minutes to remove the solvent. After removing the solvent, 1 ml of distilled water was added, and the absorption of the extracted hyperlic acid (absorbance at 2 28 nm) was measured, which was defined as oxygen activity. Note that under these conditions, the absorbance at 228 nm when no inhibitor is included is 0. The inhibition rate, which is 2 5 6, was calculated from the following formula.

Inhibition rate = (A-B)/AX t OO % A: Absorbance at 228 nm without inhibitor (0.256
) B: Absorbance at 228 nm in case of addition of inhibitor, and sample concentration at 50% inhibition rate ID.

shall be.

[Measurement of protein concentration] The protein concentration in the sample was measured by the Biulet method.
Sample concentration was calculated using bovine serum albumin as a standard protein.

(Production Example 1) Pig plasma solution 101 was P H 2 using INHcl.
．． Adjust to 0. Then add 5.6 g of pepsin.

React at 37°C for 20 hours.

Add cold methanol 381 to this (80% methanol precipitation method) and leave in a cool place overnight. The resulting precipitate was removed and concentrated under reduced pressure to give 61. Dwex5 0 w (
H') Adsorb onto the column. After thorough washing with deionized water, 2N NH. (at 0 HIOIl). Reduce the pressure to 200ml by 1tlL, Sephadex G-2
The active fraction was collected using 5-column chromatography,
Lyophilize to obtain a peptide sample. (Yield: 500 g) Test Example 1 Liquid chromatography measurement of peptides High performance liquid chromatograph; Toyo Soda HL (-80
3D type. Column: TSK gelG 3000p
wXL, φ7.8 rats X 30 cr1)
Two connected. Solution solution 20, 1% trifluoroacetic acid-45
% acetonitrile (55:45). Flow rate: 0.5
m Q/min, detector: UV-8 type, measurement wavelength 21
Qnm, preparation of calibration curve: Each standard peptide shown in Table 1 was used. Sample solution: 1g of peptide powder mixed with 10% of deionized water
100 μl of a solution obtained by diluting the solution to 0 ml and diluting the solution 50 times with an eluent was injected.

Table 1 Peptide substances used as internal standards Molecular weight ribonuclease 13,700 Cytochrome C
I2.384 Synthetic Peptide 2,200 Substance P 1,349δ-3leep Ind
ucing Peptide 849 Metynion 149 The results are shown in FIG.

The molecular weights of the peptide samples ranged from approximately 500 to 2,000.

Test Example 2 Amino acid analysis of the obtained peptide From the standard method,
The obtained peptide sample was subjected to hydrochloric acid hydrolysis, used as a sample, and analyzed using an automatic ato-amino acid analyzer.

Table 2 shows the results.

As1no acid %Asp
7.65 Thr 4.95 Set 5.43 Glu 1). 75 Pro 4.81 c1y 4.04 Ala 5.8B Cys -Val
5.60Met
-1)e
6.5BLeu
1 1. 7 5 Tyr
7.81Phe
7. 5 6Lys
8. 2 21)is
2.62Arg
7. 7 5Trp
- (Production Example 2) 1. Pig plasma 460mj provided by Pig Plasma-derived Peptide Regulator and Industry ■! PIf 2
．． After adjusting to O, 3 g of pepsin derived from porcine gastric mucosa (manufactured by Merck & Co., Ltd.) was added and enzymatically decomposed at 40°C for 7 hours. After the enzymatic decomposition was completed, the mixture was boiled for about 10 minutes to inactivate the enzyme and cooled, followed by suction filtration (Toyo Kushi Nα2) to obtain a pale yellow clear mouth fluid.

460 ml of the obtained enzymatically decomposed solution was packed into a dialysis tube (36/32 inch manufactured by Wako Pure Chemical Industries, Ltd.) and dialyzed against 51 kg of deionized water overnight in a cold room (5°C). Depressurize the external dialysis fluid]IWL, remove insoluble matter generated during IWL using a glass filter (
3G-1) to obtain purified oral fluid.

2. Purification by gel filtration column chromatography The above purified mouth liquid was concentrated under reduced pressure to 20 mJ! The mixture was closed and subjected to gel filtration chromatography four times under the conditions shown below.

Column chromatography conditions Carrier: 5ephadex G-25 (M) Column size: φ2.5 x 141. Ocm buffer: 0
．． IM phosphate buffer (PH7,0>1 fraction: volume 10.0 m 12 flow rate: 33 m'j!/h Blue dextran (Vo
, molecular weight 2 million), Vitamin V B+i
(V B+i, molecule 1) 1355) was used as a marker.

In the above gel filtration chromatography, the two fractions with strong ACEIiIl harmful activity were collected and concentrated, and lyophilized to obtain a white powder.

Experimental Example 1 Antihypertensive effect of the peptide obtained in Production Example-1 upon oral administration to rats (]) Test sample and administration method (a) The dose of the peptide obtained in Production Example-1 was 1500
The test sample was dissolved in physiological saline at a concentration of 6mj2/anial and was forcibly administered orally using a sonde.

(b) 30% chicken egg yolk solution (egg yolk is suspended in physiological saline) 5 m (1/ani+mal was orally administered).

(c) The dosage of the peptide obtained in Production Example-1 was 1500
The test sample was dissolved in 30% chicken egg yolk solution to give a concentration of 6 ml/anial, which was forcibly administered orally using a sonde.

(2) Animals used The rats used were Hos:SHR, 10-week-old male rats purchased from Experimental Animals, Inc., and preliminarily bred for one week, and then three rats were used for each test group.

(3) Blood pressure measurement Before and after administration, blood pressure was measured over time using a non-invasive tail artery blood pressure device (41 Riken Kaihatsu I'5-100).

(3) Test results The results are shown in Table 1.

1. When the post-display blood pressure value was equal to or greater than the pre-administration blood pressure value, it was indicated as 0.

As shown in Table 1, the peptide obtained in Production Example 1 had a significant hypotensive effect when administered alone orally. Furthermore, when administered orally in combination with chicken egg yolk, it exhibited an excellent blood pressure effect.

Experimental Example 2 Antihypertensive effect of the peptide obtained in Production Example-1 upon oral administration to rats (dose dependence) A 30% chicken egg yolk solution containing the peptide obtained in Production Example-1 was prepared in the same manner as in Experimental Example 1. was used to test for dose dependence. In addition, the rat is 1
Five animals were used per test group. The results are shown in Table 2.

Table 2 As shown in Table 2, when this peptide was orally administered together with chicken egg yolk liquid, it exhibited a blood pressure lowering effect depending on the dose. Moreover, the effect was excellent in persistence and did not decrease much even after 6 hours.

Experimental Example 3 Antihypertensive effect of the peptide obtained in Production Example-2 upon oral administration to rats The peptide obtained in Production Example-2 was tested in the same manner as in Experimental Example 1. As a result, the peptide obtained in Production Example-2 It was confirmed that oral administration also showed an excellent antihypertensive effect.

(Effects of the Invention) The present invention has made it possible to provide a highly safe and highly effective oral feeding composition with antihypertensive action.

Examples are shown below, and all parts in the examples mean parts by weight.

Example 1 Ice cream skim milk powder 8.0% vegetable fat
10.0 Sugar 13.0 Stabilizer
0.3 Emulsifier 0.3 Vanilla flavor 0.1 Peptide of Production Example 1 5.0 Egg yolk 7.5 Water 55.
8 Produced using a normal manufacturing method. (10.0g/pack) Example 2 Yogurt milk 64.0 Whole milk 4.0 Skim milk powder 5.0 Granulated sugar 7.0 Water
10.0 Peptide of Production Example 2 3.0 Egg yolk 7.0 Produced by normal production method. (10,0g/cu
p) Example 3 Peptide obtained in Lozenge Production Example 1 20 parts

42 Sweet potato [32.8 Tragacanth powder 5.0 Peppermint oil 0.2 Lactose 42.0 parts, Sweet potato $1
32.8 parts of powdered tragacanth, 5.0 parts of powdered tragacanth, and 0.2 parts of peppermint were mixed together, and a solution of 20.0 parts of peptide dissolved in 20.0 parts of distilled water was added thereto and thoroughly kneaded.

Next, on a glass plate sprinkled with starch, spread the above-mentioned mixture with a rolling pin to form a sheet with a thickness of about 5aii, punch it out with a mold, dry it, and apply a lozenge (0.1g / piece).

[Brief explanation of the drawing]

FIG. 1 is a liquid chromatogram of the peptide of Production Example 1.

Claims (2)

[Claims] (1) An orally ingestible composition characterized in that it contains a peptide that inhibits angiotensin converting enzyme, which is obtained by decomposing pig plasma with a proteolytic enzyme. (2) The orally ingestible composition according to claim 1, further comprising egg yolk.