HK1109563B - Protein hydrolysate with antidiabetic effect - Google Patents

Description

Protein hydrolysate with antidiabetic effect

Technical Field

The present invention relates to a composition (blood glucose increase inhibitor, insulin secretion promoter) that exhibits an action of preventing or alleviating a hyperglycemic state (blood glucose increase inhibiting action) by inducing insulin secretion or enhancing insulin sensitivity by increasing insulin receptors in a diabetic subject or a subject in a preclinical stage (borderline diabetes). Furthermore, the present invention relates to a composition for preventing or treating diseases caused by hyperglycemia, particularly diabetes and diabetic complications. In the present specification, these compositions are sometimes collectively referred to as an antidiabetic composition.

Furthermore, the present invention relates to a method for lowering the blood glucose level of a subject in a hyperglycemic state, and also relates to a method for preventing or treating diseases caused by hyperglycemia (specifically, diabetes and diabetic complications) by the action of promoting insulin secretion and the action of inhibiting blood glucose elevation.

Background

In the living body, the blood glucose level is controlled by the balance between the hypoglycemic action of insulin and the hyperglycemic action of epinephrine, glucagon, glucocorticoid and the like. Specifically, insulin inhibits glycogenolysis and gluconeogenesis in the liver to prevent the production of glucose and reduce the amount of glucose released into the blood by the liver, and at the same time, insulin increases the uptake of glucose in skeletal muscle and white adipose tissue, thereby lowering blood glucose levels. In contrast, adrenalin, glucagon, etc. promote glycogenolysis and gluconeogenesis in the liver and promote the release of glucose therefrom, thereby increasing blood glucose levels.

Diabetes is a metabolic disease in which a persistent hyperglycemic state is caused by acute or chronic decrease in insulin action, resulting in disorder of sugar metabolism, lipid metabolism, amino acid metabolism, and the like.

Diabetes is classified as either insulin-dependent or non-insulin-dependent. Dietary therapy and oral hypoglycemic agents are ineffective for treating insulin-dependent diabetes, which can only be treated by insulin due to a decrease or lack of insulin secretory capacity. In contrast, in the case of non-insulin dependent diabetes mellitus (accounting for 90% of diabetic patients), insulin is not necessarily required for its treatment, although its action is weaker than that of normal persons. Diet therapy and exercise therapy are generally used, and if the effect is not good, chemotherapy using a hypoglycemic agent may be used together.

As described above, diabetes is a disease caused by metabolic disorders resulting from a sustained hyperglycemic state, and is a very troublesome disease that may be accompanied by many complications in the eyes, kidneys, nervous system, cardiovascular system, skin, and the like. It is generally believed that these complications are eliminated when blood glucose levels are controlled around normal levels (Saishin Igaku Daijiten, 1988, p 1211, Ishiyaku Publishers Inc., Japan).

Known pharmaceutical preparations for alleviating a hyperglycemic state include insulin preparations, sulfonylurea preparations, biguanide preparations, preparations for improving insulin resistance, a-glucosidase inhibitors and the like. Insulin preparations are therapeutic drugs for insulin-dependent diabetes mellitus, and although they reliably lower blood glucose levels, they are at risk of causing hypoglycemia. Sulfonylurea preparations are drugs that lower blood glucose levels by stimulating pancreatic β -cells to promote endogenous insulin secretion. Regardless of blood glucose levels, they can cause hypoglycemic side effects due to the induction of insulin secretion. Biguanide preparations are drugs that lower blood sugar levels by inhibiting hepatic gluconeogenesis, increasing sugar consumption in skeletal muscles and the like, and inhibiting intestinal absorption of sugar, and have the advantage of causing no hypoglycemia in normal human bodies or diabetic patients. However, biguanide preparations have a problem in that relatively severe lactic acidosis is often caused. Agents that improve insulin resistance (e.g., thiazolidine derivatives and analogs) are drugs that lower blood glucose levels by potentiating the action of insulin and activating insulin receptor kinase. However, side effects such as digestive system symptoms, edema, etc. have been pointed out, and the amount of red blood cells, hematocrit and hemoglobin decreases and the amount of LDH increases (Atarashi TonyobyChiryoyaku (New diabetes Medicines), pp. 90-99, 1994, Iyaku (medicinaend drug) Journal Co., Ltd., Japan). The a-glucosidase inhibitor exhibits an effect of inhibiting postprandial increase in blood glucose level by delaying digestion and absorption of saccharides in the gastrointestinal tract, but has problems in side effects such as bloating, borborygmus, diarrhea, etc. (Joslin's diabetes mellitis, 13 th edition, page 521-522).

Therefore, it can be said that a method for effectively treating or preventing diabetes and its complications is not completely established.

Drawings

FIG. 1 shows a gel filtration chromatogram of a globulin hydrolysate (preparation example 1).

Fig. 2 shows the results (chromatograms) of the reversed-phase (acidic) chromatography performed in preparation example 2 (3).

Fig. 3 shows the results (chromatograms) of the reverse phase (neutral) chromatography performed in preparation example 2 (4).

Disclosure of Invention

An object of the present invention is to provide a pharmaceutical composition, a food composition and a feed composition, which have an insulin secretion-promoting effect or a blood glucose elevation-inhibiting effect. It is another object of the present invention to provide such a composition as a composition for treating or preventing diabetes or complications thereof.

The present inventors have made extensive studies to achieve the above object and found that when a globulin hydrolysate is administered to mice artificially induced with hyperglycemia, the blood glucose level thereof is significantly decreased and hyperglycemia is improved. Furthermore, the present inventors confirmed that the active ingredient of the globulin hydrolysate is a peptide (VVYP) contained therein, and the effect of inhibiting the increase in blood glucose is attributed to its effect of promoting insulin secretion.

Furthermore, the present inventors confirmed that the effect of globulin hydrolysate or peptide (VVYP) to suppress the increase in blood glucose level and the effect of promoting insulin secretion were not observed in subjects with normal blood glucose levels, but these effects were specifically observed in diabetic patients and borderline diabetic patients in preclinical stage.

Based on these findings, the present inventors confirmed that the globin proteolysate or peptide (VVYP) inhibits and improves hyperglycemia (lowers a blood glucose level considered to be hyperglycemic), and can be used for the prevention or treatment of diseases caused by hyperglycemia, such as diabetes and complications thereof. The present invention has been completed based on these findings.

Specifically, the present invention includes the following embodiments.

1. An antidiabetic agent comprising as an active ingredient a globulin hydrolysate or a Val-Val-Tyr-Pro peptide.

2. An agent for inhibiting elevation of blood glucose, which comprises as an active ingredient a globulin hydrolysate or a Val-Val-Tyr-Pro peptide.

3. An insulin secretion promoter comprising as an active ingredient a globulin hydrolysate or a Val-Val-Tyr-Pro peptide.

4. A special health food having a function of inhibiting elevation of blood glucose or a function of enhancing insulin secretion and preferably used with such a function, comprising as an active ingredient a globulin hydrolysate or a Val-Val-Tyr-Pro peptide.

5. A feed which has a function of inhibiting elevation of blood glucose or a function of elevating insulin secretion and is preferably used with such a function, comprising as an active ingredient a globulin hydrolysate or a Val-Val-Tyr-Pro peptide.

6. A composition for preventing or treating a disease caused by hyperglycemia, comprising as an active ingredient a globulin hydrolysate or a Val-Tyr-Pro peptide.

7. The composition for preventing or treating a disease caused by hyperglycemia according to item 6, wherein the disease caused by hyperglycemia is diabetes or a diabetic complication such as diabetic acidosis, diabetic xanthoma, diabetic muscular atrophy, diabetic ketosis, diabetic coma, diabetic gastric dysfunction, diabetic gangrene, diabetic ulcer, diabetic diarrhea, diabetic microangiopathy, diabetic uterine body sclerosis, diabetic cardiomyopathy, diabetic neuropathy, diabetic nephropathy, diabetic bulla, diabetic cataract, diabetic skin disease, diabetic sclerosis, diabetic retinopathy, diabetic lipogenic necrosis or diabetic blood circulation disorder.

8. The composition for preventing or treating a disease caused by hyperglycemia according to item 6, wherein the disease caused by hyperglycemia is type II diabetes or a complication thereof.

9. A method for inhibiting an increase in blood glucose level in a subject, comprising the step of administering an effective amount of a globin proteolysate or a Val-Tyr-Pro peptide to a subject having a disease caused by hyperglycemia or a subject in a preclinical stage of the disease.

10. The method for inhibiting an increase in blood glucose level in a subject according to item 9, wherein the disease caused by hyperglycemia is diabetes or a diabetic complication such as diabetic acidosis, diabetic xanthoma, diabetic muscular atrophy, diabetic ketosis, diabetic coma, diabetic gastric dysfunction, diabetic gangrene, diabetic ulcer, diabetic diarrhea, diabetic microangiopathy, diabetic uterine body sclerosis, diabetic cardiomyopathy, diabetic neuropathy, diabetic nephropathy, diabetic bulla, diabetic cataract, diabetic skin disease, diabetic sclerosis, diabetic retinopathy, diabetic lipogenic necrosis, or diabetic blood circulation disorder.

11. The method of inhibiting an increase in blood glucose level in a subject according to item 9, wherein the disease caused by hyperglycemia is type II diabetes or a complication thereof.

12. A method for preventing or treating a disease caused by hyperglycemia in a subject, comprising the step of administering an effective amount of a globulin hydrolysate or a Val-Tyr-Pro peptide to a subject having a disease caused by hyperglycemia or a subject in preclinical stage of the disease.

13. The method for preventing or treating a disease caused by hyperglycemia in a subject according to item 12, wherein the disease caused by hyperglycemia is diabetes or a diabetic complication such as diabetic acidosis, diabetic xanthoma, diabetic muscular atrophy, diabetic ketosis, diabetic coma, diabetic gastric dysfunction, diabetic gangrene, diabetic ulcer, diabetic diarrhea, diabetic microangiopathy, diabetic uterine body sclerosis, diabetic cardiomyopathy, diabetic neuropathy, diabetic nephropathy, diabetic bulla, diabetic cataract, diabetic skin disease, diabetic sclerosis, diabetic retinopathy, diabetic lipogenic necrosis or diabetic blood circulation disorder.

14. The method for preventing or treating a disease caused by hyperglycemia in a subject according to item 12, wherein the disease caused by hyperglycemia is type II diabetes or a complication thereof.

15. Use of a globin hydrolysate or a Val-Val-Tyr-Pro peptide in the preparation of an anti-diabetic medicament.

16. Use of a globin hydrolysate or a Val-Val-Tyr-Pro peptide in the preparation of an inhibitor of blood glucose elevation.

17. Use of a globin hydrolysate or a Val-Val-Tyr-Pro peptide in the preparation of an insulin secretion stimulator.

18. Use of a globulin hydrolysate or a Val-Val-Tyr-Pro peptide for the production of a special health food having a function of inhibiting elevation of blood glucose or a function of enhancing insulin secretion and generally used with such a function.

19. Use of a globulin hydrolysate or a Val-Val-Tyr-Pro peptide for the preparation of a feed having a function of suppressing elevation of blood glucose or a function of enhancing insulin secretion, and generally used with such a function.

20. Use of a globin hydrolysate or a Val-Val-Tyr-Pro peptide in the preparation of a composition for the prevention or treatment of a disease caused by hyperglycemia.

Detailed Description

The pharmaceutical composition, food composition and feed composition of the present invention are characterized by containing a globulin hydrolysate or peptide (VYP) as an active ingredient.

(1) Pharmaceutical composition

A blood glucose increase inhibitor, an insulin secretion promoter and a composition for preventing or treating diseases caused by hyperglycemia are included in the scope of the pharmaceutical composition of the present invention. The blood sugar increase inhibitor is characterized by containing an amount of globulin hydrolysate or peptide (VVYP) effective for preventing or improving hyperglycemia (inhibiting blood sugar increase) in diabetic patients or preclinical diabetic patients, i.e., borderline diabetic patients. The insulin secretion promoter is characterized by containing an amount of globulin hydrolysate or peptide (VVYP) effective for promoting insulin secretion in a diabetic patient or a critically ill diabetic patient. Further, the composition for preventing or treating a disease caused by hyperglycemia contains an amount of globulin hydrolysate or peptide (VVYP) effective to exert a blood sugar increase inhibitory effect or an insulin secretion promoting effect.

The globulin hydrolysate, which is one form of the active ingredient of the pharmaceutical composition of the present invention, is a hydrolysate of globulin such as hemoglobin, myoglobin, and the like. The type of animal that can be the source of globulin is not limited and many types of animals can be used, such as cattle, pigs, sheep, humans, horses, etc.

Livestock meat, fish meat and the like containing a large amount of myoglobin can be used as the globulin source.

Hydrolysis of globulin and other processes may be carried out according to the method described in WO 89/06970. Hydrolysis is typically carried out using one or more hydrolases such as acid protease, neutral protease and alkaline phosphatase.

A specific example of the method of hydrolysis of globulin is a method in which a raw material containing globulin is dispersed in water in an amount of 5 to 30% by weight (solid content), pH is adjusted to optimum conditions for a protease with an acid or a base, and the protease is added singly or in portions to allow the enzyme to react at 20 to 70 ℃ for 3 to 48 hours.

The protein hydrolysate thus obtained is used as the globulin hydrolysate having an effect of inhibiting blood glucose elevation or an effect of promoting insulin secretion of the present invention. It can be used after drying, or after adding thereto an extender such as carboxymethyl cellulose, dextrin or the like, and then drying/curing.

The peptide (VVYP), another form of the active ingredient of the pharmaceutical composition of the present invention, is contained in the aforementioned globulin hydrolysate in an amount of about 1 wt.%, and thus can be isolated/purified from the aforementioned globulin direct product. Isolation or purification of peptides (VVYP), in addition to globulins, for example, fish protein, fish meal and the like; hydrolysates of zein (zein), soy protein and similar vegetable proteins are carried out as starting materials.

The isolation or purification methods described herein can be performed in the same manner as known protein and peptide purification methods. For example, a fraction containing the peptide (VVYP) can be obtained, and the peptide (VVYP) can be isolated from the fraction, if necessary, by using salting out, dialysis, an ion exchange resin, ultrafiltration, reverse phase chromatography, or the like, or using these methods in an appropriate combination. As the reverse phase chromatography mentioned in these purification methods, it is preferable to carry out a combination of reverse phase chromatography under acidic conditions and reverse phase chromatography under neutral conditions.

The protein content of the fractions can be determined according to known protein determination methods, such as the indetrione method. The amino acid sequence of the peptide contained in the separated fraction can be determined by a known method (amino acid analysis). Thus, the presence of the target peptide of the present invention consisting of the amino acid sequence Val-Val-Tyr-Pro can be confirmed. In addition to the peptide (VVYP) isolated or purified according to the aforementioned method, a fraction containing it may be used as an active ingredient of the pharmaceutical composition of the present invention as long as the fraction exhibits an effect of inhibiting blood glucose elevation or an effect of promoting insulin secretion.

The peptide (VVYP) can be chemically synthesized according to a known peptide synthesis method. Examples of The Peptide Synthesis method include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an activated ester method, a carbonyl imidazole method, a redox method, a DCC-additive (HOMB, HOBt, HOSu) method (Schreder, Luhke, The Peptide, Vol.1, 1966, Academic Press, New York, USA; Izumiya et al, Peptide Synthesis, Maruzen Co., Ltd. (1975); etc.) and The like. These peptide synthesis methods include solid phase synthesis methods and liquid phase synthesis methods.

With respect to these peptide synthesis methods, it is preferable to protect the side chain functional groups of amino acids having side chain functional groups, such as tyrosine and threonine. Known protecting groups which can be used herein are, for example, benzyloxycarbonyl (Cbz-), tert-butoxycarbonyl (Boc-), benzyl (Bz-), etc. These protecting groups can be removed according to known methods during the production of the peptides of the invention.

The pharmaceutical composition of the invention may consist entirely of a globulin hydrolysate or peptide (VVYP). When the pharmaceutical composition is prepared in combination with other substances, the amount of the globulin hydrolysate or peptide (VVYP) is not limited as long as it sufficiently exerts an effect of inhibiting the increase in blood glucose or an effect of promoting insulin secretion. Generally, the pharmaceutical compositions of the present invention are prepared in combination with pharmacologically acceptable carriers and additives.

Examples of the carrier are an excipient, a diluent, a binder, a wetting agent, a disintegrant, a disintegration-inhibiting agent, an absorption-promoting agent, a lubricant, a solubilizing agent, a buffering agent, an emulsifying agent, a suspending agent and the like which are generally used in a dosage form for administration according to a pharmaceutical composition (formulation). Examples of additives are stabilizers, preservatives, buffers, isotonicity adjusting agents, chelating agents, pH adjusting agents, surfactants, colorants, synthetic flavors, flavoring agents, sweeteners, and the like commonly used in accordance with the dosage form in which the formulation is administered.

The unit dosage form (dosage form of pharmaceutical preparation) of the pharmaceutical composition may be appropriately selected depending on the administration route. Pharmaceutical compositions can be broadly divided into oral, pulmonary, nasal, sublingual, parenteral (injection, instillation) or the like. The pharmaceutical composition may be mixed, shaped or prepared into solid dosage forms such as tablets, pills, dispersions, powders, granules, capsules and the like according to known methods; liquid dosage forms such as solutions, suspensions, emulsions, syrups, elixirs and the like. The pharmaceutical compositions may be prepared in the form of a dry product which is, when used, rendered liquid by the addition of a suitable carrier. The pharmaceutical compositions may be prepared in any of these forms according to known methods.

The ratio of globulin hydrolysate or peptide (VYP) in the pharmaceutical composition of the present invention is not limited. Typically, the pharmaceutical composition forms a formulation containing from about 0.1 to about 80 wt.%, preferably from about 5 wt.% to about 60 wt.%, more preferably from about 20 wt.% to about 50 wt.% of the globin hydrolysate, or from about 0.001 to about 80 wt.%, preferably from about 0.01 wt.% to about 10 wt.% of the peptide (VVYP).

The dosage of the pharmaceutical composition obtained in this way may depend on the purpose of use of the pharmaceutical composition (for inhibiting blood sugar elevation, promoting insulin secretion, preventing or treating diseases caused by hyperglycemia, etc.); methods of administering the pharmaceutical compositions; the mode of administration; the age, weight, symptoms (severity of diabetes) of the patient; and other factors to make appropriate determinations. Generally, it is preferred that the amount of globin hydrolysate administered to an adult human is from about 100 to about 2000mg per day, or the amount of peptide (VYP) administered to an adult human is from about 1 to about 20mg per day.

The pharmaceutical composition is not administered in a single dose only, and may be administered in 3 to 4 doses per day. The pharmaceutical preparation of the aforementioned dosage form is administered by a route suitable for the dosage form, for example, the pharmaceutical preparation of injectable dosage form may be administered intravenously, intramuscularly, subcutaneously, intradermally, intraperitoneally or the like; pharmaceutical formulations in solid dosage form may be administered orally or the like.

As demonstrated in the following examples, the pharmaceutical composition of the present invention has an insulin secretion promoting effect due to the inclusion of globulin hydrolysate or peptide (VVYP), and exerts a hypoglycemic effect by improving the hyperglycemic state caused by the attenuation or lack of insulin action. Therefore, the pharmaceutical composition of the present invention is used as a composition for preventing or treating various diseases caused by a hyperglycemic state resulting from a weakened or deficient insulin action.

Diabetes and diabetic complications are included in these diseases. The diabetes of interest is preferably non-insulin dependent type II diabetes. The diabetic complications discussed herein refer to systemic and local diseases that develop from the progression of diabetes (preferably non-insulin dependent type II diabetes) directly or indirectly. Specific examples thereof include diabetic acidosis, diabetic xanthoma, diabetic muscular atrophy, diabetic ketosis, diabetic coma, diabetic gastric dysfunction, diabetic gangrene, diabetic ulcer, diabetic diarrhea, diabetic microangiopathy, diabetic uterine sclerosis, diabetic cardiomyopathy, diabetic neuropathy, diabetic nephropathy, diabetic bulla, diabetic cataract, diabetic skin disease, diabetic sclerosis, diabetic retinopathy, diabetic lipogenic necrosis and diabetic blood circulation disorder.

(2) Food composition

The food composition provided according to the present invention includes a special health food (including a food approved for special health use) having a function of inhibiting blood sugar elevation and preferably used with this function; special health food (including food approved for special health use) having a function of promoting insulin secretion and preferably used with this function; and special health foods (including foods approved for special health use) for preventing or treating diseases caused by hyperglycemia due to having a blood sugar elevation inhibitory action or an insulin secretion promoting action.

The special health food having a function of inhibiting blood sugar level elevation is characterized by containing a globulin hydrolysate or peptide (VVYP) in an amount effective for preventing or improving hyperglycemia (inhibiting blood sugar level elevation) in a diabetic patient or borderline diabetic patient. The packaging and advertising of the food may be accompanied by a description of the effect (blood sugar elevation-inhibiting effect). A special health food having an insulin secretion promoting function is characterized by containing a globulin hydrolysate or peptide (VVYP) in an amount effective for promoting insulin secretion in a diabetic patient or a critically ill diabetic patient. Packaging and advertising of food products may be provided with an introduction to this effect (promotion of insulin secretion). A special health food for preventing or treating diseases caused by hyperglycemia is characterized by containing a globulin hydrolysate or peptide (VVYP) in an amount effective for inhibiting the increase in blood sugar level or promoting insulin secretion. Packaging and advertising of the food product may be provided with an introduction to the effect (anti-diabetic effect).

The food composition of the invention may consist entirely of globulin hydrolysates or peptides (VVYP). When the food composition is prepared in combination with other substances, the amount of the globulin hydrolysate or peptide (VVYP) is not limited as long as it sufficiently exhibits an effect of inhibiting blood glucose elevation or an effect of promoting insulin secretion. The food compositions of the present invention may be prepared in combination with carriers and additives that may be used in food products. These food compositions include supplements and the like in the form of tablets, pills, capsules, granules, dispersions, powders, solutions (beverages) and the like prepared by mixing a globulin hydrolysate or peptide (VVYP) with food acceptable carriers and additives. The food composition of the invention also includes products containing globulin hydrolysates or peptides (VVYP), which can take the form of ordinary food and beverages.

Examples of such foods and beverages include milk beverages, lactic acid bacteria beverages, fruit juice-containing soft drinks, carbonated beverages, fruit juice beverages, plant/fruit beverages, alcoholic beverages, powdered beverages, coffee beverages, black tea beverages, green tea beverages, barley tea beverages, and the like; egg milk puddings, french buffalo puddings, juice containing puddings and the like, jellies, bavarois, yogurt and the like desserts; ice cream, frozen milk, milk ice (lactic-ice), milk ice cream, ice cream with fruit juice, soft ice cream (softcream), ice lollies (ice confections), sherbet (water ice), frozen dessert (frozenfection) and similar frozen desserts; chewing gum, bubble gum, and similar chewing gums (stick gum), sugar-coated tablet gum); chocolate beans (marble chocolate) and similar coated chocolates, strawberry chocolates, blueberry chocolates, melon chocolates (melocholates) and similar flavoured chocolates, and similar chocolates; fruit hard candies (including bonbons, butter balls, sugar beans (marbles), etc.), soft candies (including caramel, nougat, gummy candy (gummy candy), fruit marshmallow, etc.), fruit drops (drop), taffy, and the like; hard cookies (hard bisuit), crackers (okaki) (rice crackers), fresh scallop (sembei) (rice crackers), and similar baked confections (all of which are confections); clear soup (Consomm Lou soup), thick soup and the like; strawberry jam, blueberry jam, orange jam, apple jam, apricot jam, preserves, and the like; red wine and similar wines; cherry pulp, apricot, apple, strawberry and peach pulp, and similar processed fruits; ham, sausage, roasted pork and the like processed livestock meat; fish ham, fish sausages, fillets, kamaboko (steamed fish paste), chikuwa (grilled fish paste), hanpen (minced and steamed fish), satsumage (fried fish balls), datemaki (omeletwrapper), whale bacon and similar processed seafood; udon (wheat noodles), summer noodles (hiyamogi), somen (vermicelli), buckwheat (soba), Chinese noodles (Chinese noodles), spaghetti, macaroni, bifun (rice flour), harusame (vermicelli), raviolis and the like; and various types of side dishes, gluten cake (gluten cake), dried fish floss (denbu), and similar various other processed food products. The food composition of the invention is preferably in the form of a beverage or a dessert.

The amount of the active ingredient (globulin hydrolysate or peptide (VVYP)) in the food composition or the amount of the food composition to be used is not limited and may be appropriately selected within a wide range depending on the type of the food composition, the degree of desired improving effect and other factors. Although the amount of the food composition used varies depending on the type of the food composition, it may be suitably selected from the range of about 100 to about 2000mg/60kg in terms of globulin hydrolysate, or from the range of about 1 to about 20mg/60kg in terms of peptide (VYP), for a human weighing 60 kg.

The food composition of the present invention contains globulin hydrolysate or peptide (VVYP), and thus has an effect of promoting insulin secretion, and improving hyperglycemia due to impaired or deficient insulin action, thereby exhibiting a hypoglycemic effect. Therefore, the food composition of the present invention is useful for preventing or treating various diseases caused by a hyperglycemic state due to a decrease or lack of insulin action.

(3) Feed composition

The feed composition provided according to the present invention includes a feed having a function of inhibiting elevation of blood sugar; a feed having a function of promoting insulin secretion; and a feed for preventing or treating diseases caused by hyperglycemia due to the effect of inhibiting blood sugar elevation or promoting insulin secretion. These feeds can preferably be used as pet food, especially for dogs, cats and similar animals. The aforementioned feed having a function of inhibiting an increase in blood sugar is characterized by containing an amount of globulin hydrolysate or peptide (VVYP) effective for preventing or improving hyperglycemia (inhibiting an increase in blood sugar) in diabetic animals or borderline diabetic animals. The feed having the function of promoting insulin secretion is characterized by containing an amount of globulin hydrolysate or peptide (VVYP) effective for promoting insulin secretion in diabetic animals or borderline diabetic animals. The feed for preventing or treating diseases caused by hyperglycemia is characterized by containing an amount of globulin hydrolysate or peptide (VVYP) effective to exhibit an effect of inhibiting elevation of blood glucose or an effect of promoting insulin secretion.

These feed compositions include feeds prepared by mixing globulin hydrolysate or peptide (VVYP) with carriers and additives useful for feeds in the form of tablets, pills, capsules, granules, dispersions, powders, solutions, and the like. The feed composition of the invention also includes a product containing globulin hydrolysate or peptides (VVYP) in the form of a normal feed.

The amount of the active ingredient (globulin hydrolysate or peptide (VVYP)) contained in the feed composition or the dose of the feed composition is not limited and may be appropriately selected within a wide range depending on the type of the feed composition, the degree of desired improvement effect and other factors. Although the dosage of the feed composition varies depending on the type of the feed composition, it may be appropriately selected from the range of about 50 to about 1000mg/10kg in terms of globulin hydrolysate or from the range of about 0.5 to about 10mg/10kg in terms of peptide (VYP) for an animal of 10kg body weight.

Examples

The preparation examples and test examples given below illustrate the present invention in more detail, but the scope of the present invention is not limited to these examples. In the test examples below, "%" represents "weight percent (wt%)" unless otherwise specified.

Preparation of example 1Preparation of globulin hydrolysates

The method for preparing a globulin hydrolysate using bovine red blood cells is described in detail below.

To 100kg of fresh bovine red blood cells, 250 liters of water was added to conduct sufficient hemolysis. After adjusting the pH to 2.8 with phosphoric acid, 2.6X 10⁷The unit acid protease from Aspergillus niger was added to the solution and reacted at 50 ℃ for 3 hours.

After the reaction, the reaction solution was heated at 80 ℃ for 30 minutes to terminate the reaction. Thereafter, a suspension aqueous solution of calcium hydroxide was added to the reaction solution, and the pH was adjusted to 6.5. Then, 10kg of diatomaceous earth was added, and the mixture was filtered with a pressure filter. The resulting filtrate was spray-dried, thereby producing 23kg of a powdery globulin hydrolysate. The molecular weight distribution of the globulin hydrolysate was examined by gel filtration chromatography performed under the following conditions.

< gel filtration chromatography >

The instrument comprises the following steps: high performance liquid chromatograph (Shimadzu Corporation, LC-6A type)

Column: polyhydroxyyethyyl a, 5 μm, 9.4x200mm, manufactured by PolyLC inc.

Eluent: 50mM formic acid

Flow rate: 0.5ml/min

And (3) detection: UV absorption at 221nm

FIG. 1 shows a gel filtration chromatogram of a globulin hydrolysate obtained according to the above gel filtration chromatography.

Preparation of example 2Peptide fractions and purification for inhibiting elevation of blood TG levels

The peptide of the invention is obtained by the following method: (1) ion exchange, (2) ultrafiltration, (3) separation by reverse phase column chromatography under acidic conditions, and (4) separation by reverse phase chromatography under neutral conditions.

(1) Ion exchange

A10% aqueous solution containing 13.7g of the globulin hydrolyzate obtained in preparation example 1 was added to a weakly acidic cation exchange resin (Amberlite IRC)₅₀，H⁺Type, organic co., Ltd.), was stirred for 1 hour for absorption. Thereby separating the unabsorbed fraction.

(2) Ultrafiltration

The unabsorbed fraction obtained by the foregoing ion exchange was subjected to ultrafiltration using a stirring force ultrafiltration apparatus (manufactured by Advantec, UHP90K) and an ultrafiltration membrane (manufactured by Advantec, UIIH-1, molecular weight cut-off: 1000). The liquid (raffinate) remaining on the ultrafiltration membrane was collected. The fraction thus obtained was subjected to acid hydrolysis and quantified by the indetrione method. To the test tube, 1ml of hydrochloric acid having a final concentration of 6N was added per 3 to 5mg of protein, and the tube was sealed under atmospheric pressure and heated at 110 ℃ for 22 hours for acid hydrolysis. The foregoing indetrione process is carried out as follows. The pH of the hydrolyzed sample was adjusted to 5.0 with sodium hydroxide. The sample was then reacted with ninhydrin reagent dissolved in 0.2M citrate buffer (pH5.0) at 100 ℃ for 15 minutes. The absorbance at 570nm was measured. Separately, aqueous L-leucine solutions (75, 150, 225 and 300nmol/ml) were subjected to ninhydrin reactions as standard solutions. A standard curve was obtained from the measured absorbance, and the amount of amino groups corresponding to L-leucine in the sample was calculated. The quantitative results are shown in table 1. The yields based on starting globulin hydrolysate are also shown in table 1.

(3) Reversed phase (acid) chromatography

The filtrate obtained after the above ultrafiltration was subjected to reverse phase (acid) chromatography under the following conditions.

< reversed phase (acid) chromatography >

The instrument comprises the following steps: high performance liquid chromatograph (Shimadzu Corporation, LC-10A type)

Column: SuperPac Pep-S, 15 μm, 22.5X250mm, produced by Pharmacia)

Eluent: aqueous acetonitrile solution containing 0.1% trifluoroacetic acid

The linear concentration gradient of acetonitrile is 2-35%; acetonitrile concentration was varied at a rate of 1%/min

Flow rate: 5ml/min

Temperature: 40 deg.C

And (3) detection: UV absorption at 220nm

Fraction time: 53.8-54.5 minutes (fraction A)

Fig. 2 shows a chromatogram obtained by the above-described reverse phase (acid) chromatography.

The fractions thus obtained were subjected to acid hydrolysis and then quantified by amino acid analysis. To the test tube, 1ml of hydrochloric acid (final concentration of 6N HCl) was added per 3 to 5mg of protein, the test tube was sealed under atmospheric pressure, and it was heated at 110 ℃ for 22 hours for acid hydrolysis. The amino acid analysis was performed under the following conditions.

< analysis of amino acids >

The instrument comprises the following steps: high performance liquid chromatograph (Shimadzu Corporation, LC-6A type)

Column: shim-pack ISC-07/S1504Na, 7 μm, 4.0X150mm manufactured by Shimadzu corporation

Eluent: amino acid mobile phase kit (Na type), flow rate manufactured by Shimadzu Corporation: 0.3ml/min

Temperature: 55 deg.C

Reaction solution 1: assay kit OPA reagent manufactured by Shimadzu Corporation

And (3) detection: fluorescence absorption (Ex348nm, Em450nm)

The acid-hydrolyzed solution was concentrated, dried using a rotary evaporator, and agglomerated, and further dried under reduced pressure for 12 hours or more, thereby completely removing hydrochloric acid. The resultant was dissolved in 0.2M citrate buffer (pH2.2) so that the ratio of each amino acid contained was about 100 nmol/ml. The solution was filtered through a 0.45 μm filter and 10 μ l of the filtrate was packed into a column. For the standard solution, an H-type amino acid standard solution (Wako Pure chemical industries, Ltd.) mixed with 18 components was diluted 25-fold with 0.2M citrate buffer (pH2.2), and 10. mu.l of the diluted solution was packed in a column (1 nmol/10. mu.l of each amino acid). The peak area of the amino acid was calculated, and the amount of the amino acid was calculated from the ratio of the sample peak area/the standard solution peak area by analysis using Chromatopac C-R4A (Shimadzu Corporation). The results are shown in Table 1. The yield based on the globulin hydrolysate is also shown in table 1.

(4) Reverse phase (neutral) chromatography

Further, the fractions eluted and fractionated by the reverse phase (acidic) chromatography were subjected to reverse phase (neutral) chromatography under the following conditions.

< reverse phase (neutral) chromatography >

The instrument comprises the following steps: high performance liquid chromatograph (Shimadzu Corporation, LC-10A type)

Column: SuperPac Pep-S, 15 μm, 22.5X250mm, produced by Pharmacia)

Eluent: aqueous acetonitrile solution containing 20mM ammonium acetate buffer (pH6.5)

The linear concentration gradient of acetonitrile is 0-25%; the acetonitrile concentration was varied at a rate of 0.5%/min

Flow rate: 5ml/min

Temperature: 40 deg.C

And (3) detection: UV absorption at 220nm

Fractionation time: 41.7-43.2 min (fraction B) and 45.8-51.0 min (fraction C).

Fig. 3 shows a chromatogram obtained by the above-described reverse phase (neutral) chromatography. The fractions obtained were quantified as in (3) above and then identified. And calculating the amino acid composition according to the proportion of each amino acid to the total amino acid content. As a result, fraction B was VTL (Val-Thr-Leu) and fraction C was VVYP (Val-Val-Tyr-Pro). Matching the hemoglobin amino acid sequence confirms the presence of these fractions in hemoglobin. The quantitative results and the yield based on the globulin hydrolysate are shown in Table 1.

TABLE 1

Test example 1Inhibition of elevated blood glucose levels (mouse)

A glucose tolerance test was performed using the globulin hydrolysate obtained in production example 1 and the fraction C peptide (VVYP) obtained in production example 2(4) to investigate the inhibitory effect on the increase in blood glucose level.

70 male ICR mice (8 weeks old) were fasted overnight and divided into 7 groups, each consisting of 10 mice. Using an oral probe, mice of each group were gavaged with 5mg of test group 1), 10mg (test group 2) or 50mg (test group 3) of the globin hydrolysate; 1. mu.g (test 4), 2. mu.g (test 5) or 10. mu.g (test 6) of the peptide (VYP); or 0.2ml (control) of water. The globulin hydrolysate and the peptide (VVYP) were dissolved in 0.2ml of water and used. Immediately after administration, 0.2ml of glucose solution (0.25g/ml glucose in water) was gavaged in a similar manner using an oral probe. Blood was collected from the tail vein at 30, 60, 90 and 120 minutes after administration of the Glucose solution, and blood Glucose levels were determined according to the mutarotase/GOD method using a kit (Glucose CII Test Wako, Wako pure chemical Industries, Ltd.).

Blood glucose levels (AUC: area under the curve, mg/dL 120min) of the test group and the control group 120 minutes after the administration of the glucose solution are shown in Table 2. In Table 2, "percentage with respect to the control" means the ratio of the blood glucose level of the test group with respect to the blood glucose level of the control group, which is 100% (hereinafter, the same applies).

TABLE 2

Group of	Dosage (dosage/mouse)	Blood glucose level (mg/dL 120min)	Percentage relative to control
				Control group	-	1768±360	100％
Test group 1 test group 2 test group 3	Globulin hydrolyzate 5mg10mg50mg	1654±4701596±500888±204*	94％90％50％
				Test group 4 test group 5 test group 6	Peptide (VVYP) 1. mu.g 2. mu.g 10. mu.g	1537±486892±210*644±140**	87％54％40％

^*p<0.05

^**p<0.01

As can be seen from the table, the globulin hydrolysate and peptide (VVYP) significantly inhibited the increase in blood glucose level in mice in a dose-dependent manner. The peptide (VVYP) has a blood glucose lowering activity 1000 times higher than that of the globulin hydrolysate. Since the globulin hydrolysate contained the peptide (VVYP) at a ratio of about 1%, one of the active ingredients (effective ingredients) of the globulin hydrolysate having a hypoglycemic effect is presumed to be the peptide (VVYP).

Test example 2Inhibitory action on elevation of blood sugar level (Japanese)

Human (Japanese) was subjected to a sugar tolerance test (7-day drug holiday, single blind crossover test) (diabetic subjects: 12 type II diabetic patients with fasting blood glucose level of 200mg/dl or more; normal subjects: 10 normal subjects with fasting blood glucose level of 100mg/dl or less; average body weight: 68kg) using the globulin hydrolysate obtained in preparation example 1 to investigate the inhibitory effect of the globulin hydrolysate on the increase in blood glucose level.

The subjects (diabetic subjects and normal subjects) were in a starvation state due to fasting starting the evening before, and were administered 1000mg, 1500mg or 3000mg (test group) of globulin hydrolysate, or 3000mg (control group) of milk casein. Glucose solution (75g/200ml) was given immediately after administration. Blood was collected at 60, 90 and 120 minutes after the administration of the Glucose solution, and the blood Glucose level was measured according to the mutarotase/GOD method using a kit (Glucose CII Test Wako, Wako Pure chemical industries, Ltd.).

Blood glucose levels (AUC: area under the curve, mg/dL 120min) of test and control subjects 120 minutes after administration of the glucose solution are shown in Table 3.

TABLE 3

^*p<0.05

^**p<0.01

As can be seen from the table, administration of 3000mg of the globulin hydrolysate showed a statistically significant effect of inhibiting the increase in blood glucose level in both type II diabetic subjects and normal subjects. However, according to clinical applications, the globulin hydrolysate showed a more pronounced and effective effect on diabetic subjects than on normal subjects.

Thus, globulin hydrolysate is considered to significantly inhibit the increase in blood glucose in subjects with type II (non-insulin dependent) diabetes. According to the results of the above test example 1, it is believed that the main component (active ingredient) of the globulin hydrolysate is a peptide (VVYP) which is expected to have about 5000-fold higher activity of inhibiting the increase in blood glucose in patients with type II (non-insulin dependent) diabetes than the globulin hydrolysate.

Test example 3Action of increasing blood insulin level (Japanese)

12 subjects (Japanese, average body weight: 67kg) were administered with the globulin hydrolysate obtained in production example 1 in an amount of 1000 and 3000mg and a placebo (milk casein) in an amount of 1000mg for a drug holiday of 7 days (single blind crossover test). Blood samples were taken after each substance administration to determine blood insulin and blood triiodothyronine levels. Specifically, subjects fasted for more than 12 hours from the previous night. On the day of the test, blood was collected immediately before administration and every hour for 6 hours after administration to determine blood insulin and blood triiodothyronine levels. Blood Insulin levels were determined using "Glazyme Insulin-EIA TEST" (Wako Pure Chemical industries, Ltd., Osaka). The measurement of blood triiodothyronine levels was carried out by Shionogibiomedical Laboratories (Osaka) according to the CLIA method.

Blood insulin levels (mU/L.cndot.3h) and blood triiodothyronine levels (ng/L.cndot.3h) at 3 hours after administration of the test substances (1000mg and 3000mg of globulin hydrolysate and placebo) are shown in Table 4.

TABLE 4

^*p<0.05

As can be seen from the table, administration of 3000mg of globulin hydrolysate did not produce a large difference in blood triiodothyronine levels, but blood insulin levels (insulin secretion rate) were significantly increased. This fact confirms that the globulin hydrolysate has a specific promoting effect on the rate of insulin secretion in the human body (Japanese) without adversely affecting thyroid function. Further, in view of the results given in the table, it is presumed that the effect of the globulin hydrolysate shown in test example 2 on inhibiting the increase in blood glucose in a human body (japanese) is caused by its effect of promoting insulin secretion, and in view of the result of test example 1, it is presumed that the active ingredient (effective ingredient) of the globulin hydrolysate is a peptide (VVYP).

Test example 4Action of increasing blood insulin level (Western)

500, 1000 and 2000mg of the globulin hydrolysate prepared in preparation example 1 and placebo (1000mg of milk casein) were administered to 17 Western persons (7 obese subjects, 10 normal subjects, average body weight: 82kg) with a drug holiday of 7 days (single blind crossover test). Blood was collected after each substance was administered to determine blood insulin and blood glucose levels. Specifically, the subjects were given cream soup (containing 41g carbohydrate and 67g fat) along with the test substance. Blood was collected immediately before administration (0), and every hour for 4 hours after administration to measure blood insulin and blood glucose levels.

Blood Insulin levels were determined using "Glazyme Insulin-EIA TEST" (Wako Pure Chemical industries, Ltd., Osaka). Blood Glucose levels were determined according to the mutarotase/GOD method using a kit (Glucose CII Test Wako, Wako Pure Chemical Industries, Ltd., Osaka).

Blood insulin levels (AUC: area under the curve.

TABLE 5

Effect of globulin hydrolysate on blood insulin levels

As can be seen from the table, by administering the globulin hydrolysate, the blood insulin level (insulin secretion rate) of the obese subject was significantly increased, and the increase in blood glucose level was correspondingly inhibited. This effect is very pronounced when the globin hydrolysate is administered in an amount of 1000 mg. In contrast, no increase (promotion) in blood insulin level (insulin secretion rate) was observed in normal subjects. Normal subjects show a trend of decline instead.

In test example 3 conducted on the japanese subject, an administration amount of 3000mg of globulin hydrolysate showed a stronger effect of promoting blood insulin level (insulin secretion rate) than an administration amount of 1000mg of globulin hydrolysate. In contrast, in the present test example conducted on Western people, the administration amount of 1000mg of globulin hydrolysate showed a stronger effect of promoting blood insulin levels (insulin secretion-promoting effect) than the administration amount of 2000mg of globulin hydrolysate. In view of this fact, the globulin hydrolysate can be used as an effective improving or preventing composition for Japanese diabetic patients, the effect of which is expected to be better than that for Western diabetic patients.

Test example 5Blood sugar level lowering effect (Western people)

An obesity treatment plan (diet therapy, exercise therapy) was performed for 12 weeks on 30 obese subjects (age 18 to 65 years) with a BMI of 30 or more. Of these, 17 randomly selected subjects ingested a diet containing 1.5g of the globulin hydrolysate prepared in preparation example 1 once a day at home (test group). The remaining 13 subjects ingested a diet without globulin hydrolysate once a day at home (control group). After 37 weeks of this home care, the fasting blood glucose levels and body weights of the test subjects of the test group and the control group were measured, and the measurement results were compared with those measured before the home care. Blood Glucose levels were determined according to the mutarotase/GOD method using a kit (Glucose CII TestWako, Wako Pure Chemical Industries, Ltd., Osaka). Table 6 shows mean change in fasting blood glucose levels before the start of home care for test and control subjects.

TABLE 6

	Number of subjects	Weight change (kg)	Blood glucose level Change (mg/dL)
				Control group	13	-3.7±7.2	+9.0±14.1
Test group (globulin hydrolyzate administration group)	17	-3.9±9.4	-7.3±16.8

As can be seen from the table, the test subjects of both the globulin hydrolysate-administered group and the non-globulin hydrolysate-administered group (control group) showed a body weight lower than that before the start of home care. Furthermore, the group administered with globulin hydrolysate showed a significant decrease in blood glucose level. In view of the results of test example 4, it is presumed that the effect of lowering blood glucose of the globulin hydrolysate shown in this test example is caused by promotion of insulin secretion.

Test example 6Safety test

The peptide having an amino acid sequence of Val-Val-Tyr-Pro (SEQ. ID. No.1) prepared in example 2 was orally administered to male and female ICR mice in an amount of 10g/kg (maximum permissible dose) or more, and none of the cases died, thereby indicating the safety of the peptide.

Example 1Preparation of food containing peptide (VVYP)

(1) Preparing milk powder

10mg of the peptide having the amino acid sequence Val-Val-Tyr-Pro (SEQ. ID. No.1) prepared in preparation example 2 was added to 100g of infant milk powder formula, thereby obtaining milk powder having the function of promoting insulin secretion or inhibiting blood glucose elevation.

(2) Preparation of chocolate

50mg of the peptide having an amino acid sequence of Val-Val-Tyr-Pro (SEQ. ID. No.1) prepared in preparation example 2 was added to 100g of chocolate, thereby obtaining chocolate having a function of promoting insulin secretion or inhibiting blood glucose elevation.

(3) Preparation of Green tea beverage

80 kg of green tea leaves were added to 3001 hot water (80 ℃ C.) and extracted at this temperature for 4 minutes. The resulting extract was cooled and centrifuged. The clear supernatant was collected as green tea extract. To this extract was added 0.4kg of vitamin C, followed by addition of 50g of the peptide having the amino acid sequence Val-Val-Tyr-Pro (SEQ. ID. No.1) prepared in preparation example 2. Hot water was added to a final volume of 10001. The mixture is heated to 85 deg.C or higher, filled into a metal can, and subjected to retort sterilization (125 deg.C, 5 minutes) to obtain a green tea beverage.

(4) Preparation of chewing gum

Chewing gum was prepared using the following formulation.

< chewing Gum formulation >

The gum base, sugar, corn syrup and glycerin are first mixed. Then, the peptide having an amino acid sequence of Val-Val-Tyr-Pro (SEQ. ID. No.1) prepared in preparation example 2 was added, and the mixture was uniformly kneaded at 50 ℃ using a mixer. After cooling, the mixture was compression molded by a nip roll to obtain chewing gum sticks containing the peptide (VVYP) as an active ingredient in a proportion of 5mg per stick.

Example 2Preparation of a food product (1) supplement containing a globulin hydrolysate

The following ingredients were kneaded, granulated, dried and tabletted according to standard methods to obtain tablets containing globulin hydrolysate as an active ingredient, the content of the globulin hydrolysate in each tablet (200mg) being 25 wt.% (50 mg). These tablets are useful as supplements having pharmacological effects of globulin hydrolysate (antidiabetic effect, blood sugar level elevation inhibitory effect, insulin secretion promoting effect).

< tablet formulation > (Each tablet)

Example 3Preparation of feed containing the peptide of the present invention

The peptide having the amino acid sequence Val-Tyr-Pro (seq. id No.1) prepared in preparation example 2 was added to a premix containing vitamins, minerals and the like at a ratio of 0.1 wt.%. The mixture was added to a commercially available canine food at a ratio of 10 wt.% to obtain a canine food having a function of promoting insulin secretion or inhibiting blood sugar elevation.

Industrial applicability

According to the present invention, a pharmaceutical composition, a food composition and a feed composition which exert a blood sugar-lowering or insulin secretion-promoting effect due to inclusion of a globulin hydrolysate or peptide (VVYP) as an active ingredient therein can be produced. These compositions exhibit an effect of lowering hyperglycemic blood glucose levels in diabetic and borderline diabetic subjects due to their blood glucose lowering or insulin secretion promoting effects. Therefore, the composition of the present invention can be effectively used for preventing or treating diseases caused by hyperglycemia, especially diabetes and diabetic complications.

Claims (6)

1. Use of a globin hydrolysate comprising a Val-Val-Tyr-Pro peptide or a Val-Val-Tyr-Pro peptide in the manufacture of an anti-diabetic medicament.

2. Use of a globin hydrolysate containing a Val-Val-Tyr-Pro peptide or a Val-Val-Tyr-Pro peptide for the preparation of a blood glucose level increase inhibitor.

3. Use of a globin hydrolysate containing a Val-Val-Tyr-Pro peptide or a Val-Val-Tyr-Pro peptide in the preparation of an insulin secretion stimulator.

4. Use of a globulin hydrolysate containing a Val-Val-Tyr-Pro peptide or a Val-Val-Tyr-Pro peptide for the production of a special health food having a function of inhibiting blood glucose elevation or a function of improving insulin secretion and generally used with such a function.

5. Use of a globulin hydrolysate containing a Val-Val-Tyr-Pro peptide or a Val-Val-Tyr-Pro peptide for the preparation of a feed having a function of suppressing elevation of blood glucose or a function of enhancing insulin secretion, and usually used with such a function.

6. Use of a globin hydrolysate containing a Val-Val-Tyr-Pro peptide or a Val-Val-Tyr-Pro peptide in the preparation of a composition for the prevention or treatment of a disease caused by hyperglycemia.