JP3123618B2 - Novel peptide, gastric acid secretion inhibitory anti-ulcer agent and food and drink containing the peptide as an active ingredient - 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 bioactive peptide derived from human or mammalian milk or a processed product thereof. Furthermore, the present invention relates to an agent for suppressing gastric acid secretion and an anti-ulcer agent comprising the peptide as an active ingredient and a food or drink having such an action.

[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%, and the probability of recurrence is extremely high at 80% or more. In the case of recurrence, administration of an anti-ulcer drug must be started again. In other words, a patient once suffering from a 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, the secretion of gastric juice is high, 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 said to be a chronic disease can be prevented. There is a strong demand for foods that have the functionality to do so.

[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 in view of the feature that ulcer is a chronic disease, and to provide a medicament or a food or drink effective for the treatment and prevention thereof. 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 physiologically active 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 impaired peptides and the like.

Further, Vasilevskaya et al. [Vopr. Pitaniya,
4, P21-24 (1977)] reported that κ-casein glycomacropeptide (hereinafter abbreviated as GMP) obtained by enzymatically degrading κ-casein suppressed gastric acid secretion.
Guilloteau et al. [Reprod. Nutr. Develop., 27, P287-288
(1987)] reported that intravenous administration of GMP to calves did not alter gastric juice secretion. As described above, at present, no clear conclusion has been made yet on the gastric secretion inhibitory effect of GMP.

[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 a gastric acid secretion inhibitory effect and an anti-ulcer effect, and having few side effects. A further object of the present invention is to provide a gastric acid secretion inhibitor and an anti-ulcer agent or a food or beverage containing such a physiologically active peptide as an active ingredient.

[0009]

((P) represents a phosphate group)

The present invention also relates to a gastric acid secretion inhibitor and an anti-ulcer agent containing such a physiologically active peptide as an active ingredient. Furthermore, the present invention relates to a food or drink containing such a physiologically active peptide as an active ingredient and having gastric acid secretion suppression and anti-ulcer action. The bioactive peptide of the present invention can be collected from a glycomacropeptide (GMP) fraction. GMP
It has long been known that whey is released into whey during cheese production. As this raw material, it is possible to use cheese whey or a whey protein concentrate produced by ultrafiltration of cheese whey, or cheese whey or lactose mother liquor from which whey protein is precipitated and removed by heating or the like. In order to industrially produce a GMP fraction, a method disclosed in Japanese Patent Publication No. 59-27358, a method disclosed in
It is preferable to use the method described in 276542 or the method described in Japanese Patent Application No. 2-95686. A GMP fraction can also be obtained from the remaining liquid from the production of the rennet casein card by the method disclosed in JP-A-63-284199. From the GMP fraction thus obtained, an active center peptide having a gastric acid secretion inhibitory effect is isolated by ion exchange or reverse phase HPLC. Further, the peptide of the present invention may be synthesized using constituent amino acids as a raw material by 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 JP-A-2-276542, first, cheese whey, WP
C, a protein-free cheese whey or the like is obtained and adjusted to a pH of less than 4, and then subjected to ultrafiltration using a membrane having a molecular weight cut-off of 10,000 to 50,000 to obtain a permeate. And
Next, the permeate was adjusted to pH 4 or higher, and the molecular weight cut off was 5
A GMP crude fraction is obtained by desalting and concentrating using a membrane of 000 or less.

Further, the GMP crude fraction thus obtained is dissolved in a buffer solution containing 0.25 M sodium chloride at pH 8.7, and adsorbed on an anion exchange resin at pH 8.7. To elute the non-adsorbed part. Next, the adsorbed portion is eluted with a buffer containing 0.3 M sodium chloride at pH 8.7 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-mentioned amino acid sequence (see Example 1).

As described above, a large number of 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. Although the amino acid sequences of these peptides have already been elucidated, peptides containing the amino acid sequences of the present invention are not included, and all are different from the peptides of the present invention.
The peptide according to the present invention is composed of the N-terminal 1-2 of _αs2 casein.
Fourth, no physiological effects related to this peptide have been reported to date. There is no report that this peptide was isolated. 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, although the peptide of the present invention has the property of binding to an anion exchange resin, the substance described in JP-A-62-277327 is
It is a substance that is adsorbed and collected on a cation exchange resin.

[0015] From the above, 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 (hereinafter, abbreviated as intravenous injection) but also oral administration showed a gastric acid secretion inhibitory effect and an anti-ulcer effect. I found something.

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, addition, deletion or substitution, it has the same physiological activity as the above-mentioned peptide. As long as it has, 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 dragee, tablet, capsule or the like, 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 a food for preventing ulcers, the amount is preferably 0.001 mg / kg body weight / day or more, and more preferably 0.01 to 1 mg / kg body weight / day. That is, 0.001 mg / kg body weight /
Less than a day there is no noticeable increase in efficacy.

The peptide obtained as described above is
Although it is a milk-derived peptide, it has almost no antigenicity,
Less likely 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 less than 0.1 μg / kg body weight / day, and at 100 μg / kg body weight / day or more, there is no obstacle, but the effect is not particularly increased.

These may be administered once a day, or may be administered in several divided doses. 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.

[0021]

The present invention provides a novel gastric acid secretion inhibitor and a peptide having an antiulcer activity. further,
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 liters of water at 50 ° C and adjusted to pH 3.5 with concentrated hydrochloric acid. The molecular weight cut off 2
Using an ultrafiltration membrane of 000 (DDS GR61PP), 50 ° C., pressure 0.4 MPa, average permeate flow rate 5
Ultrafiltration was performed at 2.4 liter / m ² · h. When the amount of the permeated liquid reached 40 liters, 40 liters of water at 50 ° C. was added to the concentrated liquid, and ultrafiltration was continuously performed. Continuous operation was performed as described above, and a total of 160 liters of permeate was obtained. To the obtained permeate, 25% caustic soda was added to adjust the pH to 7.0, and ultrafiltration was again performed under the same conditions and with the same ultrafiltration membrane until the concentrated solution was reduced to 5 liters. Subsequently, water at 50 ° C. was added, and diafiltration was carried out with the same ultrafiltration membrane under the same conditions as before, while constantly maintaining the amount of the concentrated solution at 10 liters, followed by desalting. When the volume of the permeate reaches 80 liters by this diafiltration, the addition of water to the concentrate is stopped, and the concentrate is concentrated by ultrafiltration until the volume of the concentrate reaches 2 liters. After drying, 54 g of a crude GMP fraction containing κ-casein glycomacropeptide was obtained. As a result of an analysis by Urea SDS electrophoresis, the purity was 82%.

1 g of the crude GMP fraction obtained was
20 mM Tris / HCl buffer (pH 8.
7) It was dissolved in 50 ml and passed through an anion exchange resin DE-52 equilibrated with the same buffer. After the non-adsorbed fraction was eluted with the same buffer at 800 ml, 20 mM containing 0.3 M salt was added.
100 mg of the crude bioactive peptide fraction was eluted with 200 ml of Tris / HCl buffer (pH 8.7) and collected. 100 mg of the thus obtained crude peptide fraction was
Fractionation by reverse phase HPLC using PAK C-18 AG120 (Shiseido) yielded 40 mg of a bioactive peptide eluted at 5-7% acetonitrile. The eluate is
0.1 M NaClO ₄ /0.05 MH ₃ PO ₄ (N
aOH) (pH 9) and 100% acetonitrile. This elution pattern is shown in FIG. The bioactive peptide fraction is No. It is one.

The obtained peptide was converted to an amino acid sequencer (Model 470A) manufactured by Applied Biosystems.
In place of the analysis, it was a peptide consisting of the following amino acid sequence. Lys-Asn-Thr-Met-Glu-His-Val-Ser (P) -Ser (P) -Ser (P) -G
lu-Glu-Ser-Ile-Ile-Ser (P) -Gln-Glu-Thr-Tyr-Lys-Gln-
Glu-Lys

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 constituent amino acids were packed into a peptide synthesizer and synthesis was carried out to obtain a peptide-bound resin described in Example 1. The peptide bound to 1.63 g of 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. From the fraction eluted with 20% acetonitrile, 34 mg of a white powder of a bioactive peptide was obtained. Obtained.

Example 3 Confirmation of Inhibition of Gastric Secretion 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. The peptide obtained according to Example 1
1,1,10,100,1000 μg / kg body weight, β-
1 mg / kg body weight of lactoglobulin or physiological saline alone was intravenously injected, and 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.

[0025]

[Table 1] ──────────────────────────────────── Sample Gastric acid secretion (mEq / 4 hr) ──────────────────────────────────── 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 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))

[0026]

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.
0.001, 0.01, 0.1, 1, 10 mg / kg body weight or water alone was orally administered with the peptide obtained in Example 1 dissolved in water, and one hour later, the gastric pylorus was ligated. . Four hours later, the gastric cardia was also ligated and the stomach was excised, the volume of gastric juice collected in the stomach was measured, and the acidity was measured by titration. Table 2 shows the measurement results. As shown in the table, 0.01 mg / peptide of the present invention was used.
In the group administered over kg body weight, the amount of gastric acid secretion suppressed significantly decreased.

[0027]

[Table 2] ──────────────────────────────────── Sample Gastric acid secretion (mEq / 4 hr) ──────────────────────────────────── 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) kg body weight) 0.089 ± 0.054 water 0.287 ± 0.094 ──────────────────────────────── ──── (mean ± standard deviation (n = 8))

[0028]

Example 5 Therapeutic Effect of Gastric Ulcer by Intravenous Infusion Wistar rats (male, 8 weeks old, fasted for 16 hours)
0.5% of 70% ethanol was orally administered to each group (6 animals), and the peptide according to Example 1 immediately separated from WPC, the peptide according to Example 2 synthesized by a peptide synthesizer, or β-lactoglobulin was added to physiological saline. Was administered intravenously 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
Shown in As shown in the table, in the group to which the peptide of the present invention was administered, clear ulcer reduction was observed.

[0029]

[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──────────────────────────────度 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))

[0030]

Example 6 Example of Food for Preventing Ulcer (1) Production of Milk Drink for Preventing Ulcer The peptide of the present invention obtained in Example 1 was used to prepare a milk drink for preventing ulcer according to the following formulation. ────────────────────────────────── 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 0.18 peptide 0.01 water 84.99 Based on the ratio, 23.1 g skim milk, 1 glucose
5g, 0.06g of vitamins, 0.54g of yogurt flavor, and 0.03g of the physiologically active peptide separated from the milk of the present invention were added to 100ml of hot water heated to 60 ° C.
A solution obtained by mixing 6 g of safflower oil and 0.3 g of sugar ester (trade name: DKF160) at 60 ° C. in the solution dissolved in the above was gradually dropped and emulsified while stirring with a TK homomixer. After sterilizing by heating at 90 ° C for 5 minutes,
The product was cooled. 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 4 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.

[0032]

[Table 4] ─────────────────────────────────── Sample ulcer degree ────────乳 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─────────────────────────────────── Ulcer degree: 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))

(2) Preparation of jelly for preventing gastric ulcer The peptide of the present invention obtained in Example 1 was used to prepare jelly for preventing ulcer by the following formulation.

────────────────────────────────── Component content (wt%) ───── ───────────────────────────── sugar 15.0 prune extract 4.0 gelatin 0.5 peptide 0.05 water 80. 45 に よ り Sugar, gelatin and peptide were added to water 5
The mixture was added to 0 ml and dissolved by heating at 80 ° C., and pruned extract was added thereto and stirred, then 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 ulcer prevention effect was examined. Wistar rats (male, 8 weeks old, 6 rats in each group), which had been fasted for 16 hours, were given jelly containing the above peptide or jelly containing no peptide.
g was administered. After 1 hour, 0.5% of 70% alcohol
After 5 hours, the stomach was excised and incised in a large bay, and the mucosal surface was washed with physiological saline, and the degree of ulcer was observed. Table 4 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.

[0036]

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 filled in an ampoule under aseptic conditions to obtain an intravenous solution.

[0037]

((P) represents a phosphate group) [Brief description of the drawings]

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

[Explanation of symbols]

 1. Biologically active peptides of the present invention

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ identification code FI A23L 2/38 A61K 37/16 2/52 A23L 2/00 F (58) Fields investigated (Int.Cl. ⁷ , DB name) C07K 14/47 A23L 1/305 A61K 38/17 A61P 1/04 A23L 1/06 A23L 2/38 A23L 2/52 CA (STN) REGISTRY (STN) WPI (DIALOG) BIOSIS (DIALOG)

Claims (3)

(57) [Claims] 1. A peptide comprising the following amino acid sequence. Lys-Asn-Thr-Met-Glu-His-Val-Ser (P) -Ser (P) -Ser (P) -G
lu-Glu-Ser-Ile-Ile-Ser (P) -Gln-Glu-Thr-Tyr-Lys-Gln-
Glu-Lys ((P) indicates a phosphate group) 2. An agent for suppressing gastric acid secretion and an anti-ulcer agent comprising the peptide according to claim 1 as an active ingredient. 3. A food or drink comprising the peptide according to claim 1 as an active ingredient and having gastric acid secretion inhibitory and anti-ulcer action.