JPH0565295A - New physilogically active peptide and gastric acid secretion suppressing agent, antiulcer agent, food and drink containing the active peptide as active component - Google Patents

Abstract

PURPOSE:To provide a new physiologically active peptide originated from the human or mammalian milk or its processed product, having gastric acid secretion suppressing action, free from side effect and useful as an economically advantageous gastric acid secretion suppressing agent, antiulcer agent, ulcer-preventing food or drink, pathologic food, etc. CONSTITUTION:Raw milk, defatted milk, whey, concentrated whey protein, etc., of human or mammal such as cow, sheep and goat is used as a raw material. The raw material is dissolved in water, the pH of the solution is adjusted to 3.5 with concentrated hydrochloric acid, the solution is subjected to ultrafiltration with an ultrafiltration membrane having a fractionation molecular weight of 20,000, the pH of the filtrate is adjusted to 7.0 with 25% caustic soda solution, the obtained liquid is again subjected to ultrafiltration under the same condition to effect the desalting and the concentration and the concentrated solution is subjected to diafiltration to obtain kappa-casein glyco-macro-peptide. The product is passed through an anion exchange resin and the non-adsorbed fraction is fractionated by reversed phase high-performance liquid chromatography to obtain the objective new physiologically active peptide having the amino acid sequence of the formula a and exhibiting gastric acid secretion suppressing action.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel peptide derived from human or mammalian milk or a processed product thereof and having a physiological activity. Furthermore, the present invention relates to a medicine and a food or drink which contain an effective amount of this peptide and have gastric acid secretion inhibitory and antiulcer effects.

[0002]

2. Description of the Related Art Antiulcer agents include antacids that suppress the digestive action of gastric juice, anticholinergics, antigastrin agents, and histamine receptor antagonists that suppress gastric juice secretion itself. However, antacids such as sodium hydrogen carbonate have a short duration of action, although they are fast-acting, and induce alkalosis by long-term administration in large doses. The calcium carbonate preparation causes hypercalcemia that causes urinary tract stones, and the magnesium preparation has a drawback that diarrhea is likely to occur. In addition, anticholinergic drugs also have side effects such as dry mouth, constipation, and palpitations, so they cannot always be said to be satisfactory antiulcer drugs.
Furthermore, the mainstream peptide formulations of anti-gastrin agents are:
Since it has to be purified from animal urine and organs like urogastrone and secretin, the manufacturing cost is high,
Moreover, there is a limit to the production amount. In addition, although a histamine receptor antagonist has been recently developed, it has a problem that ulcer is likely to recur when administration is stopped. Especially in a modern society where stress is high, the lifetime morbidity rate of gastric ulcer is said to be 20%, and the probability of recurrence is very high (80% or more), and in case of recurrence, administration of the anti-ulcer drug must be restarted. That is, a patient who once suffers from gastric ulcer must continue to take an antiulcer drug, which is a pharmaceutical, on a regular basis, and even if a drug with few side effects is used, it cannot be said that there is no effect on the human body.

Under these circumstances, the gastric juice secretion inhibitory effect is high, and there is no risk of side effects, and an anti-ulcer drug that can be manufactured relatively inexpensively, or the onset and recurrence of ulcer which can be said to be a chronic disease are prevented. There is a strong demand for food materials that have the functionality to do so.

[0004]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the problems found in the conventional antiulcer agents as described above. That is, in consideration of the characteristics of ulcer as a chronic disease, the purpose of the invention is to provide a medicine or food or drink which is effective for the treatment and prevention thereof. This is obtained from relatively inexpensive raw materials, good gastric secretion inhibitory effect, is used as an anti-ulcer agent for treating ulcer, further, functional food for preventing the onset and recurrence of ulcer 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-mentioned characteristics and found that a peptide derived from milk has the above-mentioned action.

The physiologically active peptides in milk include, in addition to growth hormone, cell differentiation / growth factor, etc., calcium absorption promoting peptide, opioid peptide, angiotensin converting enzyme inhibitory peptide and the like.

In addition, Vasilevskaya et al. [Vopr. Pitaniya, 4,
21-24, (1977)] reported that a κ-casein glycomacropeptide (hereinafter abbreviated as GMP) obtained by enzymatically decomposing κ-casein suppressed the secretion of gastric acid. In 1987, Guilloteau et al. (Reprod. Nutr. Develop., 27, 287-288,
1987) reported that the intravenous administration of GMP to calves did not change the secretion of gastric juice. Thus, G
At present, no definite conclusion has yet been reached regarding the gastric juice secretion inhibitory effect of MP.

The inventors of the present invention have the following background.
Whey produced during cheese production, whey protein concentrate,
As a result of extensive studies using rats on the gastric juice secretion inhibitory effect of GMP obtained from a whey protein-containing solution such as deproteinized cheese whey, it was found that GMP itself does not have a gastric acid secretion inhibitory effect. It was found that a specific peptide sometimes mixed in the GMP fraction shows a remarkable gastric acid secretion inhibitory effect or an antiulcer effect, and the present invention has been completed.

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

A further object of the present invention is to provide a gastric acid secretion inhibitory and anti-ulcer agent or a food or drink which contains 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

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

Further, the present invention relates to a food or drink product containing such a physiologically active peptide in an effective amount to suppress gastric acid secretion and to have an antiulcer action.

The physiologically active peptide of the present invention can be collected from the GMP fraction. It has long been known that GMP is released into whey during cheese production. As this raw material, cheese whey or whey protein concentrate produced by ultrafiltration of cheese whey, or cheese whey obtained by precipitating and removing whey protein by a method such as heating, or lactose mother liquor can be used. To industrially produce the GMP fraction, Japanese Patent Publication No. 59-2735
The method disclosed in Japanese Patent Application Laid-Open No. 8-276542 or the method disclosed in Japanese Patent Application Laid-Open No. 2-276542.
It is preferable to use the method described in No. Further, from the remaining liquid for producing rennet casein card, Japanese Patent Application Laid-Open No.
The GMP fraction can also be obtained by the method disclosed in JP-A-284199. From the thus obtained GMP fraction, an active center peptide having a gastric acid secretion inhibitory effect is isolated by an ion exchange method or reverse phase HPLC. In addition, the peptide according to the present invention may be synthesized from a constituent amino acid as a raw material by a peptide synthesizer or a usual organic chemical synthesis method, or may be obtained by a genetic engineering method.

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

As an example of this preparation method, as disclosed in, for example, Japanese Patent Laid-Open No. 2-276542, cheese whey and WP obtained from these raw materials are first disclosed.
After obtaining C, deproteinized cheese whey, etc., and adjusting the pH to less than 4, it is subjected to ultrafiltration using a membrane having a cut-off molecular weight of 10,000 to 50,000 to obtain a permeate. Then, the pH of the permeate is adjusted to 4 or more, and the molecular weight cutoff of 50,
By desalting and concentrating with a membrane of less than 000 G
Obtain the MP fraction.

Further, the GMP fraction thus obtained was dissolved in a buffer solution containing 0.25 M sodium chloride at pH 8.7, adsorbed on an anion exchange resin at pH 8.7, and the same buffer solution was used. Elute the non-adsorbed portion. Next, the adsorbed portion is eluted with a buffer solution of pH 8.7 containing 0.3 M sodium chloride 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, the physiologically active peptide was analyzed by using an amino acid sequencer and found to have the above-mentioned amino acid sequence (see Example 2).

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

Although the amino acid sequences of these peptides have already been clarified, peptides containing the amino acid sequence of the present invention are not included, and any of them is different from the peptide of the present invention. Regarding the antiulcer substance in milk, there is JP-A-62-277327, but the specific structure is not clarified. Moreover, from the viewpoint of the separation method, it is considered that the physiologically active peptide of the present invention has a different structure and characteristics. That is, the peptide of the present invention has a property of binding to an anion exchange resin, but the substance described in JP-A-62-277327 is a substance recovered by adsorbing it on a cation exchange resin. From the above points, the peptide of the present invention was judged to be a novel peptide.

Animal tests were carried out for the physiological activity of this peptide, and it was found that, as shown in Examples 3 to 6, not only intravenous injection but also oral administration has a gastric acid secretion inhibitory action and an antiulcer effect. ..

In principle, the novel peptide of the present invention means a peptide having the above-mentioned amino acid sequence. Even if a 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 a gastric acid secretion inhibitor or an antiulcer agent by being used as an injection or by being administered as an oral preparation as a sugar-coated tablet, tablet, capsule or the like. Furthermore, it may 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, jelly and ice cream, and also added to sweets such as gums and candies. can do.

The peptide of the present invention is a peptide derived from a milk component, and when orally ingested, it has no adverse effect on the human body, and its ingestion amount is not particularly limited. However, when actually ingested as an anti-ulcer drug or ulcer preventive food, 0.001 mg / kg
Weight / day or more, preferably 0.01 to 1 mg / kg body weight / day is suitable. That is, the effect was not observed at 0.001 mg / kg body weight / day or less, and even if 1 mg or more was administered, there was no disorder, but the effect was not significantly increased.

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 dose of the injection solution is 0.1 μg / kg body weight / day or more, and preferably 1 to 100 μg / kg body weight / day. That is, 0.1 μg / kg body weight / day or less has no effect, and 100 μg / kg body weight / day or more has no effect, but no particular increase in effect is observed.

The effective amount in the present invention is a compound which is added to a drug or a food or drink at such a dose to produce gastric acid secretion inhibitory action and antiulcer action.

[0025]

INDUSTRIAL APPLICABILITY 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 a food, there are no side effects even when administered. (2) Since the raw material is milk, which is a food, the manufacturing cost is lower than that of conventional antiulcer agents. (3) Since it can be prepared in a larger amount than conventional anti-ulcer agents,
It can be widely used not only as a medicine but also as a food material.

The present invention will be specifically described below based on examples.

[Example 1] Separation and purification of physiologically active peptides Whey protein concentrate (Taiyo Kagaku, trade name Sanlacto N
-2) 1 kg was dissolved in 50 l of water at 50 ° C, and the pH was adjusted to 3.5 with concentrated hydrochloric acid. This has a molecular weight cutoff of 20,
000 ultrafiltration membrane (GR61PP manufactured by DDS), 50 ° C., pressure 0.4 MPa, average permeate flow rate 52.
Ultrafiltration was performed at 4 l / m ² · h. Permeate volume is 40
When it reached 1, 40 l of water at 50 ° C was added to the concentrated solution, and ultrafiltration was continuously performed. Continuous operation was carried out as described above, and a total amount of 160 l of permeate was obtained. In the obtained permeated liquid,
25% caustic soda was added to adjust the pH to 7.0, and ultrafiltration was performed again under the same conditions and the same ultrafiltration membrane as the concentrated solution became 5 l to desalt and concentrate. Subsequently, water at 50 ° C. was added, and diafiltration was performed under the same conditions and the same ultrafiltration membrane as before, while maintaining the concentrated liquid amount at 10 l, and desalting was further performed. When the amount of permeated liquid reached 80 liters by this diafiltration, stop adding water to the concentrated liquid, concentrated by ultrafiltration until the amount of concentrated liquid reached 2 l, and freeze-dried this concentrated liquid. , 54 g of κ-casein glycomacropeptide was obtained. This is Urea-SD
As a result of analysis by S electrophoresis, the purity was 82%.

1 g of the obtained GMP fraction was added to 5 mM 20 mM Tris / HCl buffer (pH 8.7) containing 0.25 M sodium chloride.
It was dissolved in 0 ml and passed through anion exchange resin DE-52 equilibrated with the same buffer. The non-adsorbed fraction is 800 with the same buffer.
After elution with 100 ml, 200 mg of a 20 mM Tris / hydrochloric acid buffer solution (pH 8.7) containing 0.3 M sodium chloride was used to elute 100 mg of the crude physiologically active peptide fraction, and the fraction was recovered. 100 mg of the crude peptide fraction thus obtained was added to CAPCELL PA.
Reversed phase HPL by KC-18 AG120 (Shiseido)
Fractionation by C was performed to obtain 40 mg of the physiologically active peptide that was eluted with 20% concentration of acetonitrile. Eluent is 0.1M
NaClO ₄ /0.05MH ₃ PO ₄ (NaOH)
(PH 9) and 100% acetonitrile were used. This elution pattern is shown in FIG. The bioactive peptide fraction is N
o. It is 3. The obtained peptide was used as an amino acid sequencer (473A by Applied Biosystems).
Type) to determine the above-mentioned amino acid sequence.

[0028]

Example 2 Peptide Synthesis of Amino Acid Sequence Based on the amino acid sequence obtained in Example 1, a peptide synthesizer (43) from Applied Biosystems was used.
0A type) was used to synthesize the peptide having the sequence described above 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 of (1) were packed in a peptide synthesizer and synthesis was performed to obtain a resin bound with the peptide of claim 1. The peptide bound to 1.63 g of resin was cleaved 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 by passing through -Rex MSZ 1-X8 yielded 150 mg of peptide. This peptide is further added to Aquapa
c RP-300 (Applied Biosystems)
Purified by reverse phase HPLC using 0.1% trifluoroacetic acid / water and 0.1% trifluoroacetic acid / acetonitrile as eluents, and 34 mg of white powder of bioactive peptide was obtained from the fraction eluted with 20% acetonitrile. Obtained.

[0029]

[Example 3] Confirmation of gastric juice secretion inhibitory effect by intravenous injection Wistar rats (male, 7 months old, 8 animals in each group) which had been fasted for 16 hours and restricted water supply for 2 hours were prepared by dissolving in saline. The peptides of the invention are 0.1, 1, 10, 10
0,1000 μg / kg body weight, β-lactoglobulin 1 mg / kg body weight, or physiological saline alone
Immediately, the pyloric region of the stomach was ligated. Four hours later, the gastric cardia was also ligated to remove the stomach, and the gastric juice accumulated in the stomach was collected and the volume was measured, and the acidity was measured by titration. The results are shown in Table 1. As shown in the table, gastric acid secretion was significantly reduced in the group to which the peptide of the present invention was administered at 1 μg / kg body weight or more.

[0030]

[Table 1] ──────────────────────────────────── Sample gastric acid secretion (mEq / 4hr) ─ ─────────────────────────────────── 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 was added to Wistar rats (male, 7 months old, 8 animals in each group) which were fasted for 16 hours and water supply was restricted for 2 hours.
0.001,0.0 of the peptide of the present invention dissolved in water
1, 0.1, 1, 10 mg / kg body weight or water alone was orally administered, and 1 hour later, the gastric pyloric region was ligated. Four hours later, the gastric cardia was also ligated to remove the stomach, and the gastric juice accumulated in the stomach was collected and the volume was measured, and the acidity was measured by titration. The measurement results are shown in Table 2. As shown in the table, gastric acid secretion was significantly reduced 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) ─ ─────────────────────────────────── 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 injection Wistar rats fasted for 16 hours (male, 8 weeks old,
0.5 ml of 70% ethanol was orally administered to each group (6 animals), and the peptide of the present invention separated from WPC, the peptide of the present invention synthesized by a peptide synthesizer, or β-lactoglobulin was immediately dissolved in physiological saline. After that, intravenous administration was performed at a dose of 0.01 mg / kg body weight.
Five hours later, the stomach was removed, a large incision was made, the mucosal surface was washed with physiological saline, and the ulcer degree was observed. The ulcer degree was divided into 6 stages according to the degree of mucosal hemorrhage lesions. The results are shown in Table 3. As shown in the table, in the group administered with the peptide of the present invention,
There was a clear reduction in ulcers.

[0034]

[Table 3] ──────────────────────────────── Sample Ulcer Degree ──────────── ───────────────────── Peptide (from WPC) 1.57 ± 1.30 Peptide (synthetic) 1.26 ± 1.19 β-lactoglobulin 3 .11 ± 1.48 Water 3.67 ± 1.38 Peptide-containing milk beverage 1.43 ± 1.21 Milk beverage 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 sores. 4 = 6-9 bleeding sores. 5 = 10 or more bleeding sores. (Average value ± standard deviation (n = 6))

[0035]

Example 6 Example of ulcer preventive food (1) Production of ulcer preventive milk drink A peptide for use in the present invention was used to prepare an ulcer preventive milk drink with the following formulation.

────────────────────────────── Component blending ratio (wt%) ────────── ───────────────────── Skim milk powder 7.7 Glucose 5.0 Safflower oil 2.0 Sugar ester 0.1 Vitamin 0.02 Yogurt flavor 18 Peptide 0.01 Water 84.99 ──────────────────────────────

Based on the above blending ratio, 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 were dissolved in 100 ml of warm water heated to 60 ° C., and then safflower oil 6 g And 0.3 g of sugar ester (trade name DKF160) at 60 ° C
The mixture obtained in (1) was gradually added dropwise with stirring using a TK homomixer to emulsify. This was heat-sterilized at 90 ° C. for 5 minutes and then cooled to 10 ° C. to obtain a product. In addition, a milk drink obtained by removing only the peptide from the above formulation table was produced in the same manner.

Next, these milk drinks were fed to rats,
The anti-ulcer effect was investigated. Wistar rats (male, 8 weeks old, 6 animals in each group) freely fed with a milk drink containing the above peptide or a milk drink produced without the peptide at the same time as fasting, 70% alcohol after 16 hours 0.
5 ml each was administered. Further, after 5 hours, the stomach was removed and a large incision was made. The mucosal surface was washed with physiological saline, and then the ulcer degree was observed. The observation results are shown in Table 3. As shown in the table, clear reduction of ulcers was observed in the group administered with the milk drink containing the peptide of the present invention.

(2) Production of gastric ulcer-preventing jelly The peptide of the present invention was used to produce a ulcer-preventing jelly by the following formulation.

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

According to the above blending ratio, sugar, gelatin and peptides were added to 50 ml of water and heated at 80 ° C. to dissolve,
Prune extract was added to this and stirred, then, transferred to a container and cooled. Also, a jelly containing no peptide of the present invention was produced in the same manner.

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

[0043]

Example 7 Ulcer Treatment Formulation (1) Capsule A 60 mg peptide of the present invention was filled in a soft capsule made of gelatin to obtain an ulcer treatment capsule.

(2) Intravenous preparation 600 μg of the peptide of the present invention was dissolved in 1 ml of sterilized physiological saline, and this was filled in ampoules under aseptic condition to obtain an intravenous injection.

[0045]

[Sequence listing] 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 1 5 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 drawings]

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

[Explanation of symbols]

 3 Bioactive peptide of the present invention

Claims (4)

[Claims] 1. A bioactive peptide Ile-Leu-Asn-Lys-Pro-Glu-Asp-Glu-Thr-His-Leu-Glu-Ala-Gln-Pro-Pro-Asp-Ala- having the following amino acid sequence: 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. A gastric acid secretion inhibitory and anti-ulcer agent, which comprises an effective amount of the physiologically active peptide according to claim 1. 4. A food or drink product containing an effective amount of the physiologically active peptide according to claim 1 and having gastric acid secretion suppression and antiulcer activity.