JPH0198445A - Food composition for oral administration - Google Patents

Abstract

PURPOSE:To provide the title composition useful as a health food, etc., for the prevention of diabetes, hyperlipemia, obesity, etc., and containing a basic peptide having a specific mol.wt. and obtained by the enzymatic decomposition of fish meat. CONSTITUTION:Meat of a fish such as sardine or hair-tail is homogenized with water, adjusted to pH 6.0 and hydrolyzed with a proteinase. The decomposed liquid is boiled to inactivate the enzyme and filtered after cooling. The filtrate is added with activated carbon and filtered again to obtain a clear crude peptide solution. The solution is concentrated under reduced pressure, added wit methanol and left standing. The obtained precipitate is dissolved in water and purified by column chromatography, etc., to obtain the objective basic peptide having a mol.wt. of 500-5,000 and high lysine content.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an oral food useful as a health food for preventing diabetes, hyperlipidemia, obesity, etc.

[Conventional technology]

Today, diseases that can be said to be brought on by modern lifestyles include diabetes, hyperlipidemia, obesity, etc., and the treatment or prevention of these diseases is important and urgent! lI! It is Jl.

Drug administration and dietary therapy are used as treatments for these diseases, but these methods are not common and have many problems in implementation.

Among bioactive peptides derived from natural products, foods or
Substances obtained from food raw materials are expected to be low-toxic and highly safe pharmacologically active substances, and their practical application is being considered.

However, since bioactive peptides are generally hydrophilic and have a relatively high molecular weight, they are poorly absorbed from the gastrointestinal tract and may lose their activity due to the action of degrading enzymes in the gastrointestinal tract. Routes are often limited to intravenous administration by injection.

[Problem to be solved by the invention]

The present inventors have previously identified diabetes and hyperlipidemia.

Focusing on the importance of daily improvement of medical conditions such as obesity (for example, intake of functional foods with pharmacological effects) and prevention of onset based on this, or mitigation of onset tendency, we are developing We have been searching for active substances that are toxic but effective when administered orally (ingested), but given the excellent properties of the fish meat-derived peptides mentioned above, especially their low toxicity, we have decided to use them for the above purpose. When we investigated the possibility of using the substance, we found that, surprisingly, although it is a peptide, it also showed an effect when administered orally, and furthermore, when administered in combination with egg yolk, the effect was enhanced. Coupled with its low toxicity and high safety, it is effective against diabetes and hyperlipidemia.

The present inventors have discovered that it is possible to provide an oral food product useful as a health food for preventing obesity and alleviating its tendency, and have completed the present invention.

[Structure of the invention]

That is, the present invention relates to an orally ingestible composition characterized by containing a basic peptide with a molecular ff of 1,500 to 5,000, which is obtained by decomposing fish meat with a proteolytic enzyme.

The present invention also relates to an orally ingestible composition characterized in that the above composition further contains egg yolk.

The preparation of the above peptides obtained by decomposing fish meat with proteolytic enzymes is described in the Bulletin of the J
apanese 5ociety of 5ci
entific Fisheries52 (11
), 1981-1984 (1986), etc., for example, as follows.

After homogenizing the muscles of fish such as sardines and hairtail with water, the pH was adjusted to 6.0, and the 0-lysis reaction solution was boiled to inactivate the enzyme. After that, cool filtration is performed. Add activated carbon to the filtrate,
Filter again to obtain a clear crude peptide solution. After concentrating this peptide solution under reduced pressure, methanol was added and the mixture was left in a cool place overnight to obtain a precipitate. After redissolving this white precipitate in water, it is purified using a cation exchange resin column and gel filtration to obtain a basic peptide with a molecular weight of 500 to 5,000 and a high lysine content.

The basic peptides derived from fish meat of the present invention as described above are usually isolated and obtained in the form of powder, and then used as is or more preferably together with a suitable non-toxic carrier for oral administration (tN absorption). It is provided for oral consumption as a composition in an appropriate shape and form.

Examples of compositions include basic peptide and egg yolk, along with pharmaceutically acceptable carriers (excipients, lubricants, binders, colorants, flavoring agents, flavoring agents, etc.), for oral administration. ! Pharmaceutical preparation forms, such as tablets (sugar-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, and sugar drinks.

The content of ACE inhibitory peptide in oral food can be selected depending on the dosage form, but is generally 1 to 100%.
% by weight.

As shown in test examples later, the oral food of the present invention having the above-mentioned structure shows that the active peptide as its active ingredient exhibits considerable ameliorating and preventive effects against the above-mentioned diseases even when administered orally, and has extremely low toxicity. Because of this, it is called diabetes and hyperlipidemia.

It can be orally administered (ingested) continuously for the purpose of preventing obesity and alleviating its tendency, and is expected to be effective when used as a health food.

When the oral food of the present invention is used for such purpose, the intake amount is suitably in the range of 1 to 500 mg/Kg body weight per day for an adult male in terms of the weight of the peptide.

The present inventors further discovered that when the active peptide and egg yolk were ingested together, the effect of -i was improved.

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 peptide to egg yolk in the oral feeding composition of the present invention is preferably in the range of 1:0.3 to 1:10.

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 pharmaceutical preparation for oral administration (e.g. tablets, etc.);
Moreover, it may be in the form of a food or a luxury item.

The content 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 is generally in the range of 5 to 100% by weight in total.

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 in the range of 5 mg to 2 g/Kg body weight of egg yolk per day.

Below, examples of the production of the above-mentioned basic peptides derived from fish meat, which are the active ingredients of the oral food of the present invention, are listed, as well as the results of animal experiments showing that the administration of these peptides alone and in combination with egg yolk is effective.

Production example 1 Basic peptide derived from sardines! After adding 51 g of deionized water to 500 g of Il-made sardine 5 ardinops melanosticta normal meat and homogenizing it, the pH was adjusted to 6.0, 10 g of proteolytic enzyme (Denazyme AP manufactured by Nagase Seikagaku Kogyo ■) was added, and 4.
Hydrolysis was carried out with stirring at 0°C for 5 hours. The decomposition reaction solution was boiled for 15 minutes to inactivate the enzyme, and then cooled.
Filtered with suction. 50 g of powdered activated carbon (Carporafine manufactured by Narita Yakuhin Fill) was added to the filtrate and after stirring, suction filtration was performed again to obtain a clear filtrate. After the enzymatically decomposed clear filtrate was concentrated under reduced pressure to IIl, methanol 101 was added, and the mixture was left in a cold place overnight, and the resulting precipitate was suction-filtered. The white precipitate was dissolved in 300 mj+ of deionized water and added to a strongly acidic cation exchange resin column (Dowex 50 WX 4 (H”
), 50~100mesh, φ4.5X2
0 cm). After thoroughly washing the column with deionized water, it was eluted with 2N-NH,OH500mj+. After removing ammonia by vacuum concentration, it was filtered through Amicon's ultrafiltration membranes YC-05 (molecular weight cutoff 500) and YM-0.
5 (molecular weight cut off 5,000)
~5,000 peptide solution was obtained and further freeze-dried to obtain 21.5 g of peptide powder.

Production Example 2 Preparation of ACE-inhibiting peptide derived from Tachiur in the same manner as in Production Example 1.
(usIepLurus) was carried out using 500 g of meat. However, instead of ultrafiltration, 5ephadex
A G-25 column was used. That is, the 2N-NH,OH eluate after treatment with the cation exchange resin column was concentrated under reduced pressure to remove ammonia, resulting in a concentrated solution of 49mj! I got it. t7
: 4 contractions? &4 ml was added to a 5 ephadex G-25 column (M
edium, φ2.3 × 140 cm), flow rate 30 m 1 / h, each fraction i 13.5 m
Gel filtration was carried out using l. Peptide fractions collected in large quantities by repeating 5ephadex G-25 column chromatography were treated with ion exchange resin again under the same conditions as above, and then freeze-dried. Peptide powder test example 1 Liquid chromatography measurement of peptides ゛ High speed Liquid chromatograph: Toyo Soda HL C-
803D type, column: TSK get G-300o
pw, φ7.8 m x 30 cm 2 pieces connected. Eluent: 0.1% trifluoroacetic acid-45% acetonitrile (55:45), flow rate: 0.5 ml
/min.

Detector: UV-8 type, measurement wavelength 210 nm. Creation of calibration curve 8 Each standard peptide shown in Table 1 was used.

Sample solution: 1 g of each basic peptide powder in 10 g of deionized water
Table 1 Peptide used as internal standard 1 Cytochrome C12384 2 Insulin 5750 3 Insulin B chain 30404 Somatostatin 163B5 Bradykinin
10526 Enkephalin 556 HP L for Sardine and Tachiwo Basic Peptide Powder
C is shown in FIGS. 1 and 2. The results show that the molecular weight of each basic peptide is in the range of about 1000 to 2000, and that the peptide consists of at least four components.

Test Example 2 Amino acid analysis of the obtained peptide From the standard method,
Each basic peptide powder was subjected to hydrochloric acid hydrolysis to prepare sample #4. As for tryptophan, it was used as a sample after alkaline decomposition with barium hydroxide.

High-speed amino acid analyzer: Hitachi model 835, ram: Hitachi custom ion exchange resin #2619F, φ2゜6+1
1+1X15CIm, column temperature: 50°C1 eluent:
0°2M sodium citrate solution. Flow rate: 0.25
mN/min, detection: ninhydrin color method and 0-
Phthalaldehyde (oPA) fluorescence method was used.

Amino acid analysis values for each basic peptide powder are shown in Table 2. Although it is thought to contain some free amino acids, the lysine content is extremely high, with a sardine basic peptide of 47.6%.
8 g / 100 g, Tachiwo Salt Aspartic Acid 4.60 2.24 Threonine 2
．． 79 1.25 Serine 3.55
1.43 Glutamic acid 4.91 1
．． 85 Proline 1.14 3.0
7 Glycine 2.39 5.17 Alanine 3.96 2.21 Valine
2.96 2.09 Methionine 0.
011 0.014 Isoleucine 2.22
1.720 Ishin 1.70 1.
03 Tyrosine 0.017 0.262 Phenylalanine 0.376 0.274 Tryptophan 0.136 0.106 Lysine
47.68 59.611 Histidine
3.54 0.732*g/100g basic peptide Next, the efficacy of the peptide obtained in the production example was tested.

Experimental Example 1 Effect of oral administration of the peptide obtained in Production Example 1 on diabetic rats (strobutocytosine-induced diabetic rats).

(1) Test method Wistar rats weighing 180g were fed with chow CA-
1 (manufactured by Nippon Talea), and after 24 hours of eating, they were subjected to experiments. Strobutocytosine (STZ, USA U
P-John) was dissolved in 0.5 tuft of citrate buffer (pH 4,5) and administered into the tail vein at a rate of 45 μ/kg. The mice were fed with CA-1 for one week after administration.

The sample had a dosage of 160 μl of the peptide obtained in Production Example-1.
The test sample was dissolved in physiological saline at a concentration of 6ae/animal and was forcibly administered orally using a sonde (peptide solution group), 30% chicken egg yolk solution (the egg yolk was suspended in physiological saline). 6d/anima I was orally administered (egg yolk group), and the test sample was 30 mg/kg so that the dose of the peptide obtained in Production Example-1 was 160 mg/kg.
6IRf/anima+ dissolved in chicken egg yolk solution was forcibly administered orally using a sonde (peptide egg yolk group), and after rearing for one week under the same conditions as the preliminary rearing, the components in the serum were examined. A control group to which STZ and the like were not administered was also examined in the same manner.

The table below shows the measured values in serum.

control 168.6 82.4
71.9 Peptide solution 535.8 7B, 9
65.2 Peptide egg yolk 515.1 Bo, 5
64.2 Egg yolk 602.5 140.3
96.4STZ 604.8 145.
As shown in 98.1 and above, the increase in blood sugar level caused by STZ was improved by approximately 10% by peptide administration, and further improvement was observed with peptide egg yolk.

Experimental Example 2 Effects on normal-fed rats and hyperlipidemic rats (hyperlipidemic rats induced by high-cholesterol diet) (1) Test method a) Effect on normal-fed rats Wistar rats weighing 230 g were fed a commercial solid diet. C
The mice were raised for acclimatization for one week with free feeding of A-1 and water.

The sample had a dosage of 150 m of the peptide obtained in Production Example-1.
A test sample dissolved in physiological saline at a concentration of 6II11/animal was orally administered by force using a probe (peptide solution group). 6 mf/animal of 30% chicken egg yolk solution (egg yolk suspended in physiological saline) was orally administered (yolk group).

The test sample was dissolved in 30% chicken egg yolk solution so that the dose of the peptide obtained in Production Example-1 was 150 μ/kg6
Id/animal was forcibly administered orally using a sonde ('
After giving birth to eggs (peptide egg yolk group) and rearing them under the same conditions as acclimatization for two weeks, the lipid components in the blood were examined. Clofibrate 200 was used as a control drug to compare the activity of the peptides.
A group administered orally (clofibrate group) at 1/kg was also tested at the same time.

b) Effect on high-cholesterol diet-induced hyperlipidemic rats Wistar rats weighing 220 g of the drug were acclimatized for one week.

An experimental diet was created by using powdered feed CA-1 as the basic diet and adding 1% cholesterol and 0.5% cholic acid to this.
Furthermore, as described in Experimental Example 2, (+)-a), each group was created and reared for 2 weeks. Then, in the blood (2) Results a) Effect on rats fed normal diet The table below shows the measured values in the serum.

Group triglyceride F Cholesterol ■/a Basic food 147.5 83.3 Peptide solution 130.2 74.5 Peptide egg yolk
120.7 69.2 Egg yolk 14
6.0 84.2 Kurofibrate 40.9
50.0 or higher, which is milder than in the clofibrate administration group, but a decreasing trend was observed in the peptide administration group, and this was even stronger in the peptide and egg yolk combination administration group.

b) Effect on hyperlipidemic rats induced by high cholesterol diet. The table below shows the measured values in serum.

Group I fatty acid cholesterol riboprotein experimental food 0.541 2
00.75 303.28 Peptide? Night/Night 0
．． 410 170.05 255.21 Peptide egg yolk 0.290 159.24 232.94 Egg yolk 0.522 197.72 301
．． 36 Kurofiburay) 0.284 154.73
257.30 and above, as in the case of normal-fed rats, a decrease in lipid components was observed by peptide administration, and a further decrease was observed by combined administration of peptide and egg yolk.

Experimental Example 3 Obese animals (genetically obese rats): Effect of peptides on Zucker “aL” (1) Test method: Using Zucker ra, t weighing 300 g,
Commercially available solid feed CA-1 was fed ad libitum. In sample 4, the dosage of the peptide obtained in Production Example-1 was 130 mg/
Ez++ji/animal, which was prepared by dissolving the test sample in physiological saline to give a total weight of 30 kg, was forcibly administered orally using a sonde (peptide egg yolk group), 6 mffi of 30% chicken egg yolk solution (1 g of egg yolk was suspended in physiological saline). /an ima
1 was orally administered (egg yolk group), and the test sample was administered in 3 doses so that the dose of the peptide obtained in Production Example-1 was 130 μ/kg.
6d/animal dissolved in 0% chicken egg yolk solution was forcibly administered orally using a sonde (peptide egg yolk group).
The animals were kept for 12 weeks and their body weights and blood components were examined.

(2) Results The measurement results are shown in the table below.

Group weight Phospholipid Cholesterol In
1tial Final mg/di
mg/diPef “Tide? Liquid 308.7 3
52.1 241.3 117.5 Peptide egg yolk 30
2.6 338.7 220.2 114.6 Egg yolk
297.2 458.1 298.1 134.
As shown in 2 and above, peptide administration improved obesity symptoms and blood composition, and combined administration of peptide and egg yolk showed even more excellent effects.

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

Examples are shown below.

In addition, all parts in the examples mean parts by weight.

Example 1 Lozenge [Composition] Peptide obtained in Production Example 1 20 parts lactose
42 sucrose
32.8 Tragacanth powder 5,0
Peppermint oil 0.2 milk $1!7
42.0 parts, sucrose 32.8 parts, tragacanth powder '5.0
1 part and 0.2 parts of peppermint were mixed, and a solution of 20.0 parts of peptide dissolved in 20.0 parts of distilled water was added thereto and kneaded well.

Next, on a glass plate sprinkled with starch, the above-mentioned mixture was spread with a rolling pin to form a sheet with a thickness of about 511 mm, punched out with a mold, dried, and a lozenge agent (1.0 g/
).

Example 2 Powder [Composition] Peptide obtained in Production Example 1 10.0 parts Chicken egg yolk (powdered) 20.0 Lactose
59.4 Magnesium stearate
0.5 Corn starch 10.0 Magnesium oxide 0.1 10.0 parts of peptide, 20.0 parts of chicken egg yolk, 10 parts of corn starch, and 0.1 minute of magnesium oxide were thoroughly mixed, and milk #M59.4 part and magnesium stearate 0
．． 5 parts were added and mixed. This mixture was divided into 1 g portions to obtain a powder.

Example 3 Ice cream skimmed milk powder 8.0% vegetable fat
1000 Sugar 13.0 Stabilizer 0.3 Emulsifier 0.3 Vanilla flavor 0.1 The peptide 5.0 Egg yolk 1O.0 Water 53.
3 Manufactured using a normal manufacturing method. (10.0g/force/heel) Example 4 Ice cream milk 64.0% whole milk
4.0 Skimmed milk powder 5.0 Granule L! 7.0 water
10.0 The peptide 3.0 Egg yolk 7.0 Figure 1 Figure 2 Procedural amendment document February 10, 1988 Patent Application No. 256807 of 1988 2, title of the invention 3, case with the person making the amendment Related Patent applicant address: 5-17-4 Sumida, Sumida-ku, Tokyo 534
5-90 Tomobuchi-cho, Bejima-ku, Osaka-shi Kanebo Co., Ltd. Patent Department Tel: (06) 921-1251 6. Column 7 for a brief explanation of the drawings in the specification subject to the amendment, contents of the amendment as shown in the attached sheet.

[Brief explanation of the drawing]

FIG. 1 is a high-performance liquid chromatography measurement diagram of a basic peptide obtained from sardines, and FIG. 2 is a high-performance liquid chromatography measurement diagram of a basic peptide obtained from redtail.

Claims (2)

[Claims] (1) An orally ingestible composition characterized by containing a basic peptide with a molecular weight of 500 to 5,000, which is obtained by decomposing fish meat with a proteolytic enzyme. (2) The orally ingestible composition according to claim (1), further containing egg yolk.