JP3108518B2 - Pharmaceutical and food additives for prevention or treatment of gastric ulcer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical and food additive for preventing or treating gastric ulcer, comprising as an active ingredient a peptide fraction having an inhibitory effect on gastric acid secretion isolated from human or mammalian milk.

[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 have immediate effects but have a short duration of action, causing alkalosis by long-term large-dose administration, and calcium carbonate preparations cause hypercalcemia, which causes urolithiasis. However, magnesium has a drawback that diarrhea is easily generated. 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. further,
Peptide preparations, which are the mainstream of antigastrin agents, must be purified from animal urine and organs, like urogastron and secretin, and therefore have high production costs and are limited in production amount.

[0003] Recently, histamine receptor antagonists have been developed, but there is a problem that the ulcer is likely to recur when the 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. When recurrence occurs, administration of 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 at all.

[0004] Under such circumstances, gastric juice secretion inhibitory effect is high, and there is no danger of side effects and the like. In addition, an antiulcer agent which can be produced 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 the provision of food materials having the functionality to perform.

[0005]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned problems of the conventional anti-ulcer drug,
This is in view of the characteristics of ulcer as a chronic disease. That is, it is an object to provide a medicine for treating ulcers or a functional food additive for preventing onset or recurrence of ulcers, which is obtained from relatively inexpensive raw materials, has a good gastric secretion inhibitory effect. I do.

Chernikov and Stan [21st International Daily Congress Vol.1 Book 2.161
(1982)] conducted an experiment in which a pepsin degradation product of kappa-casein was administered to the blood of dogs, and suggested that some of the degradation products include substances that inhibit gastric juice secretion and substances that promote gastric juice secretion. The report does not say anything about the specific experimental data. No relevant reports have been reported for some time, but Guilloteau et al. [1987] [Reproduction Nutrition Development (Reprod. Nutr.
evelop.) 27 , 287-288 (1987)] report that intravenous administration of casein macropeptide to calves did not alter gastric juice secretion. Thus, at present, no clear conclusion has yet been reached on the gastric secretion inhibitory effect of the peptide obtained by enzymatically decomposing milk protein.

[0007] In this background, the present inventors have
Whey, whey protein concentrate (W
PC), deproteinized cheese whey, lactose mother liquor, and the like. Glycomacropeptides were collected and their pharmacological activities were examined. Gastric secretion was obtained in crude fractions of glycomacropeptides obtained by ultrafiltration of these materials. The present inventors have found that they have an inhibitory action, and have completed the present invention.

[0008]

SUMMARY OF THE INVENTION The present invention relates to a food additive which can be added to a medicament and a food or drink for preventing or treating gastric ulcer, comprising a peptide fraction having a gastric secretion inhibitory effect as an active ingredient.

The raw material of the present invention includes cheese whey, a whey protein concentrate produced by ultrafiltration of cheese whey, cheese whey from which whey protein has been precipitated by a method such as heating, and lactose mother liquor. Can be used. In the present invention, glycosylated by the method described in JP-B-59-27358, JP-A-2-276542, JP-A-2-95686 or JP-A-63-284199. The macropeptide (GMP) crude fraction can be used as an active ingredient.

For example, as described in Japanese Patent Application Laid-Open No. 2-276542, raw milk, skim milk and the like of humans, cows, sheep, goats, etc. are used as raw materials, and cheese whey, WPC, PH 4 protein cheese whey etc.
After adjusting to less than 10,000, ultrafiltration is performed using a membrane having a cut-off molecular weight of 10,000 to 50,000 to obtain a filtrate. Then, transfer this permeate to pH
It is adjusted to 4.0 or more and concentrated using a membrane having a molecular weight cut-off of 10,000 to 50,000 to obtain a crude GMP fraction. Alternatively, as described in Japanese Patent Application No. 3-19112, a pretreatment for bringing the raw material into contact with an anion exchange resin and eluting the adsorbed fraction thereof may be performed. The crude GMP fraction may be adsorbed on an anion exchange resin, and the adsorbed fraction may be eluted with a buffer having an ionic strength of 0.25 or more, and the resulting eluate may be used. This eluate is a crude solution of the peptide. At this time, the adsorption to the anion exchange resin may be performed by dissolving the crude GMP fraction in a buffer solution of pH 8.7 containing 0.2 M sodium chloride and adsorbing this to the anion exchange resin at pH 8.7. . Then, the non-adsorbed portion is eluted using this buffer, and the adsorbed fraction is then buffered with an ionic strength of 0.25 or more,
For example, elution is carried out with a buffer solution containing 0.25M or more, desirably pH8.7 containing 1M salt, to obtain a crude peptide fraction. At this time, Q-Sepharose, DEAE-Sepharose, QAE-Sephadex, QAE-Toyopearl, etc. are used as the anion exchange resin, and Tris-HCl buffer, ethanolamine buffer, phosphate buffer, etc. Is used.

The crude peptide fraction thus obtained may be used as an anti-ulcer agent in a solution state, but is usually dried by spray drying or freeze drying.
It may be used in a powder state. In addition, since these crude peptide fractions are stable to heat, it is more desirable to carry out a sterilization step before the drying step.

The crude peptide fraction obtained as described above can be used as an oral anti-ulcer agent such as sugar-coated tablets, tablets or capsules. In addition, various foods and drinks as food additives, for example, soft drinks, fruit juice drinks,
By adding it to fermented beverages, jellies, ice creams, etc., functional foods and drinks having a preventive or therapeutic effect on ulcers can also be obtained. Furthermore, it can be added to sweets such as gums and candies.

The crude peptide fraction of the active ingredient in the present invention is a peptide derived from milk, which has no adverse effect on the human body when taken orally, and its intake is particularly limited. There is no. However, when it is actually taken orally as an anti-ulcer agent or a food for treating or preventing ulcer, 0.1 to 1000 mg / kg body weight / day is appropriate,
Desirably, it is 1 to 100 mg / Kg body weight / day. That is,
No effect is observed at 0.1 mg / Kg body weight / day or less, and there is no side effect, but no remarkable increase in effect is observed even at 1000 mg or more. The present invention is characterized in that a large amount of the peptide can be continuously administered for a long period of time.

Further, among the active ingredients in the present invention, GM
The peptide crude fraction obtained by further purifying the crude P fraction with an anion exchange resin can also be used as a drug such as an injection or other preparations. These crude fractions are said to have little antigenicity, even though they are milk-derived peptides, and are unlikely to cause allergic symptoms and the like, and the dosage as an injection is 0.01 to 100 mg / Kg body weight. / Day, preferably about 0.1 to 10 mg / kg body weight / day. In other words, there is no effect at 0.01 mg / Kg body weight / day or less, and at 100 mg / Kg body weight / day or more, no side effect is observed and there is no obstacle, but the effect is not particularly increased.

[0015] In the case of oral administration and injection, it may be administered once a day, or may be administered in several divided doses.

[0016]

The effects of the antiulcer agent or the food additive having such an effect according to the present invention are summarized as follows. (1) Since the peptide is derived from milk which is usually taken as food, there is no concern about side effects due to administration. (2) Since milk, which is a food, is used as a raw material, the production cost is very low as compared with conventional anti-ulcer agents. (3) Because it can be prepared in a larger amount than conventional anti-ulcer drugs,
It can be widely used as a food additive as well as a pharmaceutical by adding it to various foods and beverages.

Hereinafter, the present invention will be specifically described based on Examples and Reference Examples.

[0018]

Reference Example 1 This example shows a method for preparing a peptide fraction having an inhibitory effect on gastric acid secretion in the present invention. Whey protein concentrate (Taiyo Chemical, trade name Sunlac N-
2) Dissolve 1 kg in 50 liters of water at 50 ° C, and add concentrated hydrochloric acid to pH 3.
After adjusting to 5, an ultrafiltration membrane (DD
Ultrafiltration using S, GR61pp (0.4MPa pressure)
, Average permeation flow rate 52.4 l / m ² · h). Permeate volume is 4
When the volume reached 0 liters, 40 liters of water at 50 ° C. was added to the concentrated solution, followed by continuous ultrafiltration to obtain 160 liters of permeate. Add 25% sodium hydroxide solution to this permeate
The pH was adjusted to 7.0, and ultrafiltration was performed again using the same ultrafiltration membrane until the concentrated solution was reduced to 5 liters, followed by desalting and concentration. Then 50 ℃
Of diafiltration was carried out under the same conditions as before while constantly maintaining the amount of the concentrated solution at 10 liters by adding water. When the amount of the permeate reached 80 liters, the addition of the concentrate to the concentrate was stopped. After the concentrate was concentrated to 2 liters, the concentrate was freeze-dried to obtain 54 g of a crude GMP fraction. This fraction was analyzed by urea-SDS electrophoresis
82%.

[0019]

[Reference Example 2] 1.0 g of crude GMP fraction obtained in Reference Example 1
Was dissolved in 20 ml of 20 mM ethanolamine buffer (pH 8.7) containing 0.2 M salt, passed through 200 ml of Q-Sepharose Fast Flow Anion Exchange Resin, and further buffered with the same buffer.
Wash the resin with 600 ml to elute the non-adsorbed fraction. Next, the eluted fraction is collected by passing through 300 ml of 20 mM ethanolamine buffer at pH 8.7 containing 0.3 M salt. The eluted fraction is freeze-dried and the crude peptide fraction of the present invention 50 mg
Get.

[0020]

Example 1 This example shows the effect of inhibiting gastric secretion in an intravenous administration test of an antiulcer substance according to the present invention. [Test method] 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,
GMP crude fraction according to Reference Example 1 dissolved in physiological saline
0.01, 0.1, 1, 10, 100 mg / Kg body weight, β-lactoglobulin at 1 mg / Kg body weight, or physiological saline alone were intravenously injected, and the gastric pylorus was immediately ligated. Four hours later, the pyloric part of the stomach was also ligated to remove the stomach, and the gastric juice collected in the stomach was collected to measure the volume, and the acidity was measured by titration. As shown in Table 1, in the group to which the GMP crude fraction was administered at 0.1 mg / Kg body weight or more, the amount of gastric acid secretion was significantly reduced.

[0021]

[Table 1]

[0022]

Example 2 This example shows the gastric secretion inhibitory effect in an oral administration test of the antiulcer substance of the present invention. [Test Method] Wistar rats (male, 7 months old, 8 animals in each group), which had been fasted for 16 hours and restricted for 2 hours, were given 0.
The crude GMP fraction of Reference Example 1 dissolved in 2 ml of water was
0.1, 1, 10, 100, 1000 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. As shown in Table 2, in the group to which the GMP crude fraction was administered at 1 mg / Kg body weight or more, the amount of gastric acid secretion was significantly reduced.

[0023]

[Table 2]

[0024]

Example 3 This example shows the effect of treating gastric ulcer in an intravenous administration test of the antiulcer substance according to the present invention. [Test method] Wistar rats (male,
(8-week-old, 6 animals in each group) were orally administered with 0.5% of 70% ethanol, and immediately, 1 mg / kg body weight of crude peptide according to Reference Example 2 and 1 mg of β-lactoglobulin dissolved in physiological saline.
/ Kg body weight or saline alone was injected intravenously. 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. As shown in Table 3, in the group to which the crude peptide fraction was administered, clear reduction of ulcer was observed.

[0025]

[Table 3] Ulcer degree: 0 = No ulcer is 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. (Mean ± standard deviation (n = 6)

[0026]

Example 4 This example shows an example in which the anti-ulcer substance of the present invention was used in a food for preventing ulcer. (1) Production of Milk Beverage for Ulcer Prevention Using the GMP crude fraction of Reference Example 1 as a food additive, a milk beverage for ulcer prevention was prepared according to the following formulation.

[0027]

[Table 4]

Based on the above composition, 23.1 g of skim milk, 15 g of glucose, 0.06 g of vitamins, 0.54 of yogurt flavor
g, and 3 g of the crude GMP fraction according to Reference Example 1 at 60 ° C.
Safflower oil 6 in a solution dissolved in 100 ml of warm water
g and sugar ester (brand name DKF160) 0.3g at 60 ℃
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, G
A milk drink without MP alone was similarly produced. Next, these milk drinks were given to rats, and the anti-ulcer effect was examined.

[Test Method] A milk drink to which a food additive consisting of the crude GMP fraction of Reference Example 1 was added at the same time as the start of fasting,
Alternatively, Wistar rats (male, 8 weeks old, 6 rats in each group) were allowed to freely drink the milk beverage produced except for the GMP crude fraction, and 16 hours later, 0.5 ml of 70% alcohol 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. As shown in Table 2, in the group to which the milk beverage to which the food additive composed of the GMP crude fraction was added was clearly reduced in the ulcer.

(2) Production of Jelly for Preventing Gastric Ulcer A jelly for preventing ulcer was produced from the crude GMP fraction of Reference Example 1 by the following formulation.

[0031]

[Table 5]

With the above composition, sugar, gelatin, and the GMP crude fraction of Reference Example 1 were used as food additives, and 50 ml of water was added thereto.
Was added thereto, and the mixture was heated and dissolved at 80 ° C., and pruned extract was added thereto, followed by stirring, and then transferred to a container and cooled. G
Jelly containing no MP crude fraction was similarly prepared.

Next, these jellies were given to rats,
The effect of preventing gastric ulcer was examined. [Test method] Wistar rats (male,
8 weeks old, 6 mice in each group), 2 g of jelly containing the above crude GMP fraction or jelly containing no crude GMP fraction
Each dose was administered. One hour later, 0.5 ml of 70% alcohol was administered, and 5 hours later, the stomach was excised and incised into the bay, and the mucosal surface was washed with physiological saline, and the ulcer degree was observed. Table 3
As shown in the figure, in the group to which the jelly containing the crude GMP fraction was administered, clear reduction of ulcer was observed.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-5-246882 (JP, A) JP-A-5-65295 (JP, A) JP-A-5-262793 (JP, A) JP-A-5-262793 210647 (JP, A) JP-A-62-277327 (JP, A) JP-A-2-276542 (JP, A) Chemical Abstracts, Vol. 92, No. 3 (1980), Abst. No. 16362e Chemical Abstracts, Vol. 91, No. 11 (1979), Abst. No. 84171w (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 38/00-38/58 A23L 1/305 A61K 35/20 BIOSIS (STN) CA (STN) MEDLINE (STN)

Claims (4)

(57) [Claims] 1. Whey, whey of human or mammalian milk
Protein concentrate (WPC), deproteinized cheese whey or lactose mother
The aqueous solution is permeated through an ultrafiltration membrane with a molecular weight cut-off of 10,000 to 50,000 at a pH below 4 and a molecular weight cut-off of 50,000 at a pH of 4 or more.
Using the following ultrafiltration membrane was concentrated and desalted, and further adsorbed to an anion exchange resin, the more sodium chloride 0.25M adsorbed fraction
Peptide obtained by elution with a buffer containing
An agent for preventing or treating gastric ulcer comprising a crude fraction as an active ingredient. 2. The peptide is a glycomacropeptide.
The agent for preventing or treating gastric ulcer according to claim 1. 3. Whey, whey of human or mammalian milk
Protein concentrate (WPC), deproteinized cheese whey or lactose mother
Solution with a molecular weight cut off of 10,000 to 50,000
Permeation through ultrafiltration membrane, molecular weight cut off at pH 4 or higher 50,000
Demineralize and concentrate using the following ultrafiltration membrane.
Adsorbed on an exchange resin, and the adsorbed fraction
Peptide obtained by elution with a buffer containing
A food additive having a gastric ulcer preventive or therapeutic action, comprising a crude fraction as a gastric acid secretion inhibitory component. 4. The food additive having a prophylactic or therapeutic effect on gastric ulcer according to claim 3, wherein the peptide is a glycomacropeptide.