JP2907603B2 - Novel physiologically active peptide, gastric acid secretion inhibitor containing the active peptide as an active ingredient, anti-ulcer agent, and food and drink - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel peptide having a physiological activity derived from human or mammalian milk or processed products thereof. Furthermore, the present invention relates to a medicament, food or drink having an inhibitory effect on gastric acid secretion and an anti-ulcer effect containing an effective amount of this peptide.

[0002]

2. Description of the Related Art Antiulcer agents include antacids for suppressing gastric juice digestion, anticholinergic agents for suppressing gastric secretion itself, antigastrin agents, and histamine receptor antagonists. However, antacids, such as sodium bicarbonate, are quick-acting but have a short duration of action, and induce alkalosis by long-term large dose administration. Calcium carbonate preparations cause hypercalcemia which causes urinary calculi, and magnesium preparations have a drawback that diarrhea is likely to occur. In addition, anticholinergic agents also have side effects such as dry mouth, constipation, and palpitations, and cannot always be said to be satisfactory antiulcer agents.
Furthermore, the mainstream peptide preparations of anti-gastrin agents are:
Since it must be purified from animal urine and organs, like urogastron and secretin, production costs are high,
There is also a limit on the amount of production. Recently, histamine receptor antagonists have been developed, but there is a problem that ulcers are likely to recur when administration is stopped. Particularly in a stressful modern society, the lifetime morbidity of gastric ulcer is said to be 20%, the probability of recurrence is extremely high at 80% or more, and when recurrence occurs, administration of the anti-ulcer drug must be started again. In other words, a patient once suffering from gastric ulcer must regularly take an antiulcer drug, which is a pharmaceutical, and even if a drug with few side effects is used, it cannot be said that there is no effect on the human body.

[0003] Under these circumstances, gastric juice secretion is highly effective, and there is no danger of side effects. In addition, an antiulcer agent which can be produced relatively inexpensively, or the onset or recurrence of ulcer, which can be regarded as a chronic disease, is prevented. There is a strong demand for food materials having the functionality to perform.

[0004]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems of the conventional anti-ulcer drug. That is, the present invention has been made for the purpose of providing a medicament or a food or drink which is effective for the treatment and prevention of ulcers in consideration of the characteristics of the disease as a chronic disease. This is obtained from relatively inexpensive raw materials, has a good gastric secretion inhibitory effect, is used as an anti-ulcer agent for treating ulcers, and is a functional food for preventing the onset and recurrence of ulcers. Alternatively, it is intended to provide an active ingredient that can be used as a pathological diet. The present inventors have searched for an active ingredient having the above characteristics and found that a peptide derived from milk has the above-mentioned action.

[0005] Physiologically active peptides in milk include, in addition to growth hormone and cell differentiation / growth factors, calcium absorption promoting peptides, opioid peptides, angiotensin converting enzyme inhibitory peptides and the like.

Further, Vasilevskaya et al. [Vopr. Pitaniya, 4,
21-24, (1977)] reported that kappa-casein glycomacropeptide (hereinafter abbreviated as GMP) obtained by enzymatically decomposing kappa-casein inhibits gastric acid secretion, but in 1987 Guilloteau et al. (Reprod. Nutr.Develop., 27, 287-288,
1987) reported that intravenous administration of GMP to calves did not alter gastric juice secretion. Thus, G
At present, no clear conclusion has yet been reached on the gastric secretion inhibitory effect of MP.

[0007] In this background, the present inventors have
Whey produced during cheese production, whey protein concentrate,
After extensive studies using rats to examine the gastric secretion inhibitory effect of GMP obtained from a whey protein-containing solution such as deproteinized cheese whey, it was found that GMP did not have a gastric acid secretion inhibitory effect, but that GMP was prepared. The present inventors have found that a specific peptide which is sometimes mixed in the GMP fraction exhibits a remarkable gastric acid secretion inhibitory effect or an antiulcer effect, and has accomplished the present invention.

[0008] That is, an object of the present invention is to provide a novel bioactive peptide having gastric acid secretion suppression and anti-ulcer action, and having few side effects.

It is a further object of the present invention to provide a gastric acid secretion inhibitor and an anti-ulcer agent or a food or beverage containing an effective amount of such a physiologically active peptide.

[0010]

The present invention resides in a physiologically active peptide having the following amino acid sequence. Ile-Leu-Asn-Lys-Pro-Glu-Asp-Glu-Thr-His-Leu-Glu-Ala-Gln-Pro-Pro-Asp-Ala-Ser-Ala-Gln-Phe-Ile-Arg-Asn- Leu-Gln-Ile-Ser-Asn-Glu-Asp-Leu-Ile-Ser-Lys-Glu-Gln-Ile-Val-Ile-Arg

[0011] The present invention also relates to a gastric acid secretion inhibitor and an anti-ulcer agent containing an effective amount of such a physiologically active peptide.

Further, the present invention relates to a food or drink having an inhibitory effect on gastric acid secretion and an anti-ulcer effect, which contains an effective amount of such a physiologically active peptide.

The physiologically active peptide of the present invention can be collected from a GMP fraction. It has long been known that GMP is released into whey during cheese production. As this raw material, it is possible to use cheese whey, a whey protein concentrate produced by ultrafiltration of cheese whey, cheese whey from which whey protein has been precipitated and removed by heating or the like, or lactose mother liquor. For industrial production of GMP fraction, Japanese Patent Publication No. 59-2735
No. 8, JP-A-2-276542, or Japanese Patent Application No. 2-95686.
It is preferable to use the method described in the above item. Further, the remaining liquid from the production of rennet casein card was obtained from
The GMP fraction can also be obtained by the method disclosed in JP-A-284199. An active center peptide having a gastric acid secretion inhibitory effect is isolated from the GMP fraction thus obtained by ion exchange or reverse phase HPLC. The peptide according to the present invention may be synthesized from constituent amino acids using a peptide synthesizer or a general organic chemical synthesis method, or may be obtained by a genetic engineering technique.

That is, the physiologically active peptide of the present invention comprises:
Raw milk, skim milk, whey or whey protein concentrate (WP) of humans or mammals such as cows, sheep and goats
It is prepared using C) as a raw material.

As an example of this preparation method, for example, as disclosed in Japanese Patent Application Laid-Open No. 2-276542, cheese whey obtained from these raw materials, WP
C, a protein-free cheese whey or the like is obtained and adjusted to a pH of less than 4, followed by ultrafiltration using a membrane having a molecular weight cut-off of 10,000 to 50,000 to obtain a permeate. Then, the permeate was adjusted to pH 4 or higher, and the molecular weight cutoff was 50,
By desalting and concentrating using a membrane of
Obtain the MP fraction.

Further, the GMP fraction thus obtained is dissolved in a pH 8.7 buffer solution containing 0.25 M sodium chloride, adsorbed on an anion exchange resin at pH 8.7, and the same buffer solution is used. Elute the non-adsorbed part. Next, the adsorbed portion is eluted with a buffer solution of pH 8.7 containing 0.3 M salt to obtain a crude peptide fraction. The crude peptide fraction is fractionated by reverse phase HPLC to obtain a purified bioactive peptide (see Example 1).

In the present invention, analysis of this physiologically active peptide using an amino acid sequencer revealed that the peptide had the above amino acid sequence (see Example 2).

Numerous physiologically active peptides in milk, such as growth hormone, cell differentiation / growth factor, calcium absorption promoting peptide, opioid peptide, and angiotensin converting peptide, are known as described above.

Although the amino acid sequences of these peptides have already been elucidated, peptides containing the amino acid sequence of the present invention are not included, and all are different from the peptides of the present invention. Japanese Patent Application Laid-Open No. 62-277327 discloses an anti-ulcer substance in milk, but no specific structure has been disclosed. Moreover, from the viewpoint of the separation method, it is considered that the structure and properties are different from those of the physiologically active peptide of the present invention. That is, the peptide of the present invention has the property of binding to an anion exchange resin, but the substance described in JP-A-62-277327 is a substance that is adsorbed and recovered on a cation exchange resin. From the above points, the peptide of the present invention was determined to be a novel peptide.

An animal test was conducted on the physiological activity of this peptide. As shown in Examples 3 to 6, not only intravenous injection but also oral administration revealed that the peptide had an inhibitory effect on gastric acid secretion and an antiulcer effect. .

The novel peptide of the present invention means a peptide having the above-mentioned amino acid sequence in principle. Even if the peptide has several amino acids added, deleted or substituted, it has the same physiological activity as the above-mentioned peptide. As long as it is included in the novel peptide of the present invention. The physiologically active peptide of the present invention can be used as an injection or as an oral preparation as a sugar-coated tablet, tablet, or capsule, and used as a gastric acid secretion inhibitor or an antiulcer agent. Furthermore, it can be used as an anti-ulcer food material by adding it to various foods and drinks, such as soft drinks, fruit juice drinks, fermented drinks, and jelly and ice cream, and also to confectionery such as gum and candy. can do.

The peptide of the present invention is a peptide derived from a milk component. When taken orally, it has no adverse effect on the human body, and there is no particular limitation on the amount of the peptide. However, when it is actually taken orally as an anti-ulcer agent or an ulcer-preventive food, 0.001 mg / kg
Body weight / day or more, desirably 0.01 to 1 mg / kg body weight / day is appropriate. That is, no effect is observed at a dose of 0.001 mg / kg body weight / day or less, and no significant increase in the effect is observed even though administration of 1 mg or more does not cause any problem.

The peptide obtained as described above is
Although it is a milk-derived peptide, it has little antigenicity and is unlikely to cause allergic symptoms. The dosage of the injection solution is 0.1 μg / kg body weight / day or more, preferably 1 to 100 μg / kg body weight / day. That is, there is no effect at 0.1 μg / kg body weight / day or less, and no effect is observed at 100 μg / kg body weight / day or more, although there is no obstacle at all.

The effective amount in the present invention refers to a dose which is added to a medicine or food or drink in such an amount as to give a gastric acid secretion inhibitory effect and an anti-ulcer effect.

[0025]

The present invention provides a novel peptide having a gastric acid secretion inhibitory action and an antitumor action. Furthermore, the physiologically active peptide of the present invention has the following effects. (1) Since it is a peptide derived from milk which is usually taken as food, there is no side effect even if administered. (2) Since milk, which is a food, is used as a raw material, the production cost is lower than that of a conventional anti-ulcer agent. (3) Because it can be prepared in a larger amount than conventional anti-ulcer drugs,
It can be widely used not only as a pharmaceutical but also as a food material.

Hereinafter, the present invention will be specifically described based on examples.

Example 1 Separation and purification of a physiologically active peptide Whey protein concentrate (manufactured by Taiyo Chemical Co., trade name: Sunlac N)
-2) 1 kg was dissolved in 50 l of water at 50 ° C. and adjusted to pH 3.5 with concentrated hydrochloric acid. This was compared with the molecular weight cutoff of 20,
000 ultrafiltration membrane (GR61PP from DDS), 50 ° C., pressure 0.4 MPa, average permeate flow rate
Ultrafiltration was performed at 4 l / m ² · h. Permeate volume is 40
When the concentration reached 1 l, 40 l of water at 50 ° C. was added to the concentrate, and ultrafiltration was continuously performed. Continuous operation was performed as described above, and a total of 160 l of permeate was obtained. In the obtained permeate,
25% caustic soda was added to adjust the pH to 7.0, and ultrafiltration was performed again under the same conditions and with the same ultrafiltration membrane until the concentrated solution became 5 liters and desalted and concentrated. Subsequently, water at 50 ° C. was added, diafiltration was carried out with the same ultrafiltration membrane under the same conditions as before, and the salt was further desalted, while always keeping the amount of the concentrated solution at 10 l. When the volume of the permeate reached 80 l by this diafiltration, the addition of water to the concentrate was stopped, and the concentrate was concentrated by ultrafiltration until the volume of the concentrate reached 2 l, and the concentrate was freeze-dried. Thus, 54 g of κ-casein glycomacropeptide was obtained. This is Urea-SD
As a result of analysis by S electrophoresis, the purity was 82%.

[0027] 1 g of the obtained GMP fraction was added to a 20 mM Tris / HCl buffer (pH 8.7) containing 0.25 M sodium chloride.
It was dissolved in 0 ml and passed through an anion exchange resin DE-52 equilibrated with the same buffer. The unadsorbed fraction was 800
After elution with 100 ml, 100 mg of the crude bioactive peptide fraction was eluted and collected with 200 ml of 20 mM Tris / HCl buffer (pH 8.7) containing 0.3 M salt. 100 mg of the thus-obtained crude peptide fraction was subjected to CAPCELL PA
Reverse phase HPL by K C-18 AG120 (Shiseido)
By fractionation with C, 40 mg of a bioactive peptide eluted at 20% acetonitrile concentration was obtained. The eluate is 0.1M
NaClO ₄ /0.05 MH ₃ PO ₄ (NaOH)
(PH 9) and 100% acetonitrile. This elution pattern is shown in FIG. Bioactive peptide fraction is N
o. 3. The obtained peptide was used as an amino acid sequencer (473A) of Applied Biosystems.
And the amino acid sequence described above was determined.

[0028]

Example 2 Synthesis of Peptide of Amino Acid Sequence Based on the amino acid sequence obtained in Example 1, a peptide synthesizer of Applied Biosystems (43)
0A), the peptide having the above sequence was synthesized by the t-Boc method. That is, 0.5 mmol of Boc-L
-Cln-O-CH ₂ -PAM resin and each 2mmol
The synthesis was carried out by filling the constituent amino acids into a peptide synthesizer to obtain a peptide-bound resin according to claim 1. The peptide bound to 1.63 g of the resin was excised with trifluoromethanesulfonic acid in the presence of thioanisole and ethanedithiol, precipitated with diethyl ether, dissolved with 10% acetic acid, and replaced with 10% acetic acid. Basic anion exchange resin Bio
Purification through -Rex MSZ 1-X8 gave 150 mg of peptide. This peptide is further converted to Aquapa
c RP-300 (Applied Biosystems)
Was purified using 0.1% trifluoroacetic acid / water and 0.1% trifluoroacetic acid / acetonitrile as eluent, and 34 mg of a white powder of a bioactive peptide was obtained from the fraction eluted with 20% acetonitrile. Obtained.

[0029]

Example 3 Confirmation of Gastric Secretion Inhibitory Effect by Intravenous Injection Wistar rats (male, 7 months old, 8 rats in each group), which had been fasted for 16 hours and restricted for 2 hours, were dissolved in saline. 0.1, 1, 10, 10
0,1000 μg / kg body weight, β-lactoglobulin 1 mg / kg body weight, or intravenous saline only,
The gastric pylorus was immediately ligated. Four hours later, the gastric cardia was also ligated, and the stomach was removed. The gastric juice collected in the stomach was collected, the volume was measured, and the acidity was measured by titration. Table 1 shows the results. As shown in the table, in the group to which the peptide of the present invention was administered at 1 μg / kg body weight or more, the amount of gastric acid secretion was significantly reduced.

[0030]

[Table 1] Sample Gastric acid secretion (mEq / 4hr)ペ プ チ ド Peptide (0.1 μg / kg body weight) 0.281 ± 0. 105 (1 μg / kg body weight) 0.108 ± 0.088 (10 μg / kg body weight) 0.096 ± 0.052 (100 μg / kg body weight) 0.066 ± 0.092 (1 mg / kg body weight) 0.058 ± 0.049 β-lactoglobulin (1 mg / kg body weight) 0.291 ± 0.070 physiological saline 0.327 ± 0.089 ──────────────────── ──────────────── (mean ± standard deviation (n = 8))

[0031]

Example 4 Confirmation of Gastric Secretion Inhibitory Effect by Oral Administration 0.2 ml of Wistar rats (male, 7 months old, 8 rats in each group) which had been fasted for 16 hours and water supply was restricted for 2 hours.
Of the peptide of the present invention dissolved in
1, 0.1, 1, 10 mg / kg body weight or water alone was orally administered, and one hour later, the pyloric part of the stomach was ligated. Four hours later, the gastric cardia was also ligated, and the stomach was removed. The gastric juice collected in the stomach was collected, the volume was measured, and the acidity was measured by titration. Table 2 shows the measurement results. As shown in the table, gastric acid secretion significantly decreased in the group to which the peptide of the present invention was administered at 0.01 mg / kg body weight or more.

[0032]

Table 2 Sample Gastric acid secretion (mEq / 4hr)ペ プ チ ド Peptide (0.001 mg / kg body weight) 0.276 ± 0. 105 (0.01 mg / kg body weight) 0.132 ± 0.096 (0.1 mg / kg body weight) 0.106 ± 0.081 (1 mg / kg body weight) 0.071 ± 0.046 (10 mg / kg body weight) 0.089 ± 0.054 Water 0.287 ± 0.094 ─────────────────────────────────── (Average value ± standard deviation (n = 8))

[0033]

Example 5 Therapeutic Effect of Gastric Ulcer by Intravenous Infusion Wistar rats (male, 8 weeks old, fasted for 16 hours)
0.5 ml of 70% ethanol was orally administered to each group (6 animals), and the peptide of the present invention immediately separated from WPC, the peptide of the present invention synthesized by the peptide synthesizer, or β-lactoglobulin was dissolved in physiological saline. After that, the solution was intravenously administered at a dose of 0.01 mg / kg body weight.
Five hours later, the stomach was removed and an incision was made on the stomach. The mucosal surface was washed with physiological saline, and the degree of ulcer was observed. The ulcer degree was divided into six stages according to the degree of mucosal hemorrhagic lesion. Table 3 shows the results. As shown in the table, in the group to which the peptide of the present invention was administered,
There was a clear reduction in ulcers.

[0034]

[Table 3] ──────────────────────────────── Sample ulcer degree ─────────── ───────────────────── Peptide (derived from WPC) 1.57 ± 1.30 Peptide (synthetic product) 1.26 ± 1.19 β-lactoglobulin 3 .11 ± 1.48 water 3.67 ± 1.38 peptide-containing milk drink 1.43 ± 1.21 milk drink 2.98 ± 1.45 peptide-containing jelly 1.87 ± 1.54 jelly 3.01 ± 1 .55───────────────────────────────── Degree of ulcer: 0 = No ulcer was found in the gastric mucosa. 1 = Redness only. 2 = 1 bleeding erosion. 3 = 2-5 bleeding erosions. 4 = 6-9 bleeding erosions. 5 = 10 or more bleeding erosions. (Average value ± standard deviation (n = 6))

[0035]

Example 6 Example of Food for Preventing Ulcer (1) Production of Milk Drink for Preventing Ulcer A milk drink for preventing ulcer was prepared by using the peptide of the present invention and the following composition.

────────────────────────────── Ingredients Compounding ratio (wt%) ───────── ───────────────────── Skim milk powder 7.7 Glucose 5.0 Safflower oil 2.0 Sugar ester 0.1 Vitamins 0.02 Yogurt flavor 0.0 18 peptide 0.01 water 84.99──────────────────────────────

Based on the above proportions, skim milk 23.1
g, glucose 15 g, vitamins 0.06 g, yogurt flavor 0.54 g, and physiologically active peptide 0.03 g separated from the milk of the present invention in 100 ml of warm water heated to 60 ° C. in a solution of 6 g of safflower oil. 0.3 g of sugar ester (trade name DKF160) at 60 ° C
The mixture obtained in the above was gradually dropped and emulsified while stirring with a TK homomixer. This was sterilized by heating at 90 ° C. for 5 minutes, and then cooled to 10 ° C. to obtain a product. In addition, a milk beverage in which only the peptide was removed from the above composition table was produced in the same manner.

Next, these milk drinks were given to rats,
The anti-ulcer effect was examined. Wistar rats (male, 8 weeks old, 6 rats in each group) were allowed to freely drink a milk drink containing the peptide or a milk drink prepared without the peptide at the same time as the start of fasting. To 0.
Each 5 ml was administered. Five more hours later, the stomach was removed and an incision was made in the bay, and the mucosal surface was washed with physiological saline, and the degree of ulcer was observed. Table 3 shows the observation results. As shown in the table, in the group to which the milk beverage containing the peptide of the present invention was administered, clear reduction of ulcer was observed.

(2) Production of jelly for preventing gastric ulcer A jelly for preventing ulcer was produced from the peptide of the present invention by the following formulation.

─────────────────────────── Component content (wt%) ──────────── ─────────────── Sugar 15.0 Prune extract 4.0 Gelatin 0.5 Peptide 0.05 Water 80.45 ────────────── ─────────────

According to the above mixing ratio, sugar, gelatin and peptide are added to 50 ml of water and dissolved by heating at 80 ° C.
After adding prune extract to this and stirring, it was transferred to a container and cooled. Jelly containing no peptide of the present invention was similarly produced.

Next, these jellies were given to rats,
The effect of preventing gastric ulcer was examined. Wistar rats (male, 8 weeks old, 6 rats in each group) which had been fasted for 16 hours were administered with 2 g of the above-mentioned peptide-containing jelly or peptide-free jelly. After 1 hour, add 70% alcohol 0.5
The stomach was further removed 5 hours later, the stomach was removed, a large incision was made, the mucosal surface was washed with physiological saline, and the degree of ulcer was observed. Table 3 shows the observation results. As shown in this table, in the group to which the jelly containing the peptide of the present invention was administered, clear reduction of ulcer was observed.

[0043]

Example 7 Ulcer Treatment Formulation (1) Capsule A capsule for treating ulcer was obtained by filling 60 mg of the peptide of the present invention into a soft capsule made of gelatin.

(2) Intravenous Injection 600 μg of the peptide of the present invention was dissolved in 1 ml of sterile physiological saline, and this was aseptically filled into an ampule to obtain an intravenous solution.

[0045]

[Sequence list] SEQ ID NO: 1 Sequence length: 42 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ile Leu Asn Lys Pro Glu Asp Glu Thr His Leu Glu Ala Gln Pro Pro Asp Ala 15 10 15 Ser Ala Gln Phe Ile Arg Asn Leu Gln Ile Ser Asn Glu Asp Leu Ile Ser Lys 20 25 30 35 Glu Gln Ile Val Ile Arg 40 42

[Brief description of the drawings]

FIG. 1 shows an elution pattern of a physiologically active peptide of the present invention in reverse phase HPLC.

[Description of Signs] 3. Biologically active peptide of the present invention

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C07K 14/47 ZNA A61K 38/00 CA (STN) REGISTRY (STN)

Claims (4)

(57) [Claims] 1. A bioactive peptide having the following amino acid sequence: Ile-Leu-Asn-Lys-Pro-Glu-Asp-Glu-Thr-His-Leu-Glu-Ala-Gln-Pro-Pro-Asp-Ala- Ser-Ala-Gln-Phe-Ile-Arg-Asn-Leu-Gln-Ile-Ser-Asn-Glu-Asp-Leu-Ile-Ser-Lys-Glu-Gln-Ile-Val-Ile-Arg 2. The physiologically active peptide according to claim 1, which is derived from human or mammalian milk or a processed product thereof. 3. An agent for suppressing gastric acid secretion and an anti-ulcer agent, comprising an effective amount of the physiologically active peptide according to claim 1. 4. A food or drink having an inhibitory effect on gastric acid secretion and an anti-ulcer effect, comprising an effective amount of the physiologically active peptide according to claim (1).