JP5041780B2 - Liver disorder improving composition - Google Patents

Description

  The present invention relates to a composition for improving liver damage. More specifically, the present invention relates to a composition for improving liver damage that has a preventive or ameliorating effect on liver damage caused by hepatitis virus, alcohol, stress, drugs or immune abnormalities.

  With the recent westernization of dietary habits, there is a marked increase in the number of patients with liver damage. It is known that hepatic disorders may cause abnormal lipid metabolism due to excessive intake of fat and cholesterol, which causes a decrease in liver function. The liver plays a central role in detoxification and substance metabolism as a biological chemical factory, and works as a major organ with various functions. However, it is well known that the liver can be damaged either acutely or chronically by drug side effects, environmental toxins, alcohol and viral infringement. In order to maintain the normal function of the living body, there has been a wide demand for the development of an effective liver disorder ameliorating agent having no side effects.

The globin proteolysate is a peptide mixture that has been conventionally used for the purpose of suppressing the increase in neutral fat in the blood (see, for example, Patent Document 1), but it has an action to improve liver damage. Until not known at all.
WO89 / 006970 International Publication

  The object of the present invention is to provide a composition for improving liver damage as described above. In particular, an object of the present invention is to provide a composition for improving liver damage that has a preventive or ameliorating effect on liver damage caused by hepatitis virus, alcohol, stress, drugs or immune abnormalities.

  The inventors of the present invention have made extensive studies to solve the above-mentioned problems. As a result, the globin proteolysate that has been conventionally used for suppressing the increase in neutral fat in blood has an action to improve liver damage. It has been confirmed that there is a preventive or therapeutic effect on liver damage caused by the headline, particularly hepatitis virus, alcohol, or drugs. The present invention has been completed based on such findings, and is a composition for improving liver damage characterized by comprising a globin proteolysate as an active ingredient.

Specifically, the present invention includes the following embodiments:
Item 1. A composition for improving liver damage comprising a globin proteolysate as an active ingredient. The composition for improving liver damage includes a pharmaceutical composition and a food composition.
Item 2. Item 2. The hepatic disorder improving composition according to Item 1, wherein the hepatic disorder is a hepatic disorder caused by hepatitis virus, alcohol, stress, drugs or immune abnormalities.
Item 3. Item 3. The composition for improving liver damage according to item 1 or 2, which is a preventive or therapeutic agent for alcoholic liver damage.
Item 4. Item 3. The liver injury-improving composition according to Item 1 or 2, which is a health food containing an effective amount of a globin proteolysate that exhibits an effect of improving liver injury.
Item 5. Item 5. The hepatic disorder improving composition according to any one of Items 1, 2, and 4, wherein the composition is a health food exclusively for humans who have or are concerned about hepatic disorder.
Item 6. Item 6. The hepatic disorder improving composition according to any one of Items 1, 2, 4, and 5, which is a health food used for the purpose of preventing or treating alcoholic liver disorder.

  The composition for improving liver damage according to the present invention is characterized by exhibiting a remarkable liver damage improving action by using a globin proteolysate as an active ingredient. According to the composition for improving liver damage of the present invention, by having such an action for improving liver damage, it is possible to prevent or improve a decrease in liver function caused by liver damage.

(1) Liver injury improving composition The liver injury improving composition provided by the present invention includes a pharmaceutical composition and a food composition as described later. Here, the pharmaceutical composition targeted by the present invention is intended for patients with hepatic disorders, particularly patients with hepatic disorders caused by hepatitis virus, alcohol, stress, drugs or immune abnormalities, or patients concerned. In addition, pharmaceuticals used exclusively for the purpose of preventing (suppressing liver function deterioration) or treating (improving liver function) are included. In addition, the food composition targeted by the present invention is intended only for humans with liver damage, particularly those with liver damage caused by hepatitis virus, alcohol, stress, drugs or immune abnormalities, or those who are concerned. Health foods used for the purpose of prevention (suppression of liver function decline) or improvement (improvement of liver function) are included.

  The globin proteolysate which is an active ingredient of the composition for improving liver damage of the present invention is a hydrolyzate of globin proteins such as hemoglobin and myoglobin.

  The origin of this globin protein is not particularly limited, and examples thereof include blood of animals and fish containing a large amount of hemoglobin, or animal and fish meat (livestock meat, fish meat) containing a large amount of myoglobin. In addition, the kind of animal which is a supply source of globin protein is not specifically limited, A cow, a pig, a sheep, a human, a horse etc. can be illustrated widely.

  Operations relating to the hydrolysis of globin protein and the like can be performed according to the method described in International Publication WO89 / 06970. Hydrolysis is usually carried out using one or more hydrolases of acid protease, neutral protease or alkaline protease. Specifically, in order to hydrolyze globin protein, first, a globin protein-containing material such as blood or meat is dispersed in water so as to be 5 to 30% by weight (as a solid content), and protease or Examples thereof include a method of adjusting to an optimum pH, adding a protease at once or sequentially, and reacting the enzyme at a temperature of 20 to 70 ° C. for 3 to 48 hours. The proteolysate obtained in this way can be used as it is or dried and used as a globin proteolysate. Furthermore, an appropriate amount of a bulking agent such as carboxymethylcellulose or dextrin is added to the proteolysate and dried and solidified. It can also be used as a globin proteolysate in a state.

(2) Pharmaceutical composition As an embodiment of the composition for improving liver damage of the present invention, a pharmaceutical composition can be mentioned.

  The pharmaceutical composition targeted by the present invention includes a preventive or therapeutic agent for liver damage caused by hepatitis virus, alcohol, stress, drugs or immune abnormalities, among liver disorders.

  The pharmaceutical composition of the present invention may consist only of the above-mentioned globin proteolysate, but is usually prepared with a pharmaceutically acceptable carrier or additive in addition to the globin proteolysate.

  Here, as the carrier, excipients, diluents, binders, disintegrants, disintegration inhibitors, absorption enhancers, lubricants, solubilizers that are usually used according to the administration form of the pharmaceutical composition (formulation) , Buffers, emulsifiers, suspending agents and the like.

  As additives, stabilizers, preservatives, buffers, tonicity agents, chelating agents, pH adjusters, surfactants, coloring agents, flavoring agents, flavoring agents that are usually used according to the dosage form of the preparation And sweeteners.

  In addition, the dosage unit form (pharmaceutical preparation form) of such a pharmaceutical composition can be appropriately selected depending on the administration route, and these are largely oral agents, pulmonary agents, nasal agents, sublingual agents, parenteral agents. (Injection, infusion) These are formulated and molded into solid dosage forms such as tablets, pills, powders, powders, granules, and capsules; liquid dosage forms such as solutions, suspensions, emulsions, syrups, and elixirs according to conventional methods. Or can be prepared. Further, it may be prepared as a dry product that can be made liquid by adding an appropriate carrier before use (for example, dry syrup or an injection for preparation at the time of use). Any of these can be prepared according to a conventional method. These various forms of pharmaceutical preparations can be administered through an appropriate administration route according to the form. For example, a pharmaceutical preparation having an injection form can be administered by intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal administration, and the like, and a solid preparation can be administered by oral administration or the like.

  The ratio of the globin proteolysate blended in the pharmaceutical composition of the present invention is not particularly limited, and can be usually about 0.1 to 80% by weight, and the pharmaceutical composition of the present invention is within such a range. It can be prepared in a pharmaceutical form containing a globin proteolysate.

  The dosage of the pharmaceutical composition thus obtained includes the purpose of the pharmaceutical composition (prevention or treatment of liver damage), the method of administration of the composition, the dosage form, the type and symptom of the liver damage to the patient, age It is appropriately selected according to the weight and the like. In general, it is preferable to administer the globin proteolysate, which is an active ingredient, within a range of about 0.1 to 10 g, preferably about 0.5 to 5 g per day for an adult. In addition, the said administration does not necessarily need to be once a day, It is also possible to divide and administer in 3-4 times a day.

  The pharmaceutical composition of the present invention exhibits an effect of improving liver damage due to a globin proteolysate contained as an active ingredient as shown in the following experimental examples. The liver disorder to be targeted here is particularly caused by hepatitis virus, alcohol, stress, drug or immune abnormality, and the pharmaceutical composition of the present invention is caused by such hepatitis virus, alcohol, stress, drug or immune disorder. It can be effectively used for prevention (suppression of liver function decline) or treatment (improvement of liver function) for a patient having a severe liver disorder or a patient concerned about it.

(3) Food composition As another aspect of the composition for improving liver damage of the present invention, a food composition can be mentioned.
The food composition provided by the present invention includes health foods (including functional foods and foods for specified health use) imparted with an action for improving liver damage.

  Here, the health food means food with the purpose of health, health maintenance and promotion in a more positive sense than normal food. It should be noted that foods for specified health use (including foods for specified specified health use) that have been given an action to improve liver damage are characterized by containing an effective amount of globin proteolysate that prevents or ameliorates liver damage. In the present invention, the food packaging package or advertisement can be described with respect to its action effect (liver disorder improving action) and can be differentiated from other foods. It is a preferred embodiment.

  The food composition of the present invention may comprise only the globin proteolysate as long as it contains the globin proteolysate as described above, as long as it contains an effective amount that exhibits an effect of improving liver damage. In addition to the globin proteolysate, it is prepared with a carrier or additive that can be used as a food, and with other food materials.

  In the food composition, the above-mentioned globin proteolysate is mixed with food-acceptable carriers and additives as necessary, together with tablets, pills, capsules, granules, powders, powders, or solutions (drinks) And the like, which are prepared in various dosage forms as described above with respect to the pharmaceutical composition, and the like, for the purpose of preventing or improving liver damage. The food composition of the present invention is prepared by adding the globin proteolysate to a general food (in other words, by using the globin proteolysate as one of food ingredients). Includes health foods (food and drinks) aimed at preventing or improving liver damage.

  Examples of such foods and drinks include milk drinks, lactic acid bacteria drinks, fruit juice soft drinks, soft drinks, carbonated drinks, fruit juice drinks, vegetable drinks, vegetable / fruit juice drinks, alcoholic drinks, powdered drinks, coffee drinks, tea drinks, green tea drinks, barley tea Beverages such as beverages; custard pudding, milk pudding, souffle pudding, puddings such as pudding with fruit juice, desserts such as jelly, bavaroa and yogurt; ice cream, ice milk, lacto ice, milk ice cream, ice cream with fruit juice and Frozen confectionery such as soft ice cream, ice candy, sherbet, ice confectionery; gums such as chewing gum and bubble gum (plate gum, sugar-coated granule gum); strawberry chocolate, blueberry chocolate, melon chocolate, etc. in addition to coated chocolate such as marble chocolate s wind Chocolates such as chocolate with added candy; hard candy (including bonbon, butterball, marble, etc.), soft candy (including caramel, nougat, gummy candy, marshmallow, etc.), caramels such as drop, toffee; hard biscuits, Baked confectionery such as cookies, rice crackers, rice crackers (and confectionery); soups such as consomme soup and potage soup; jams such as strawberry jam, blueberry jam, marmalade, apple jam, apricot jam, prasab; red wine etc. Fruit wine; Syrup pickled cherries, apricots, apples, strawberries, peaches, etc .; processed meat such as ham, sausage and grilled pork; fish ham, fish sausage, fish meat surimi, salmon, bamboo rings, hampen, fried Satsuma Fisheries training such as Date roll, whale bacon Products; it can be cited other, various processed foods such as various delicatessen; udon, hiyamugi, somen, buckwheat, buckwheat middle reeds, spaghetti, macaroni, rice vermicelli, noodles such as vermicelli and wonton. Beverages and confectionery are preferred.

  The amount of the active ingredient (globin proteolysate) to be contained in the food composition can be appropriately adjusted according to the intake amount of the food composition. The amount of food composition intake varies depending on the type of food composition, the type and severity of symptoms of the target human liver disorder, and other conditions. The ratio of the decomposition product is about 0.1 to 10 g, preferably 0.5 to 5 g.

  The food composition of the present invention does not necessarily need to be taken once a day, and can be taken 3 to 4 times a day.

  That is, the ratio of the globin proteolysate blended in the food composition is usually from the range of about 0.1 to 80% by weight so that the daily intake of the globin proteolysate is within the above range. It can be appropriately selected and set.

  The food composition of the present invention improves liver damage caused by globin proteolysate contained as an active ingredient, particularly hepatopathy caused by hepatitis virus, alcohol, stress, drugs or immune abnormalities as shown in the following experimental examples. Demonstrate the effect. Therefore, the food composition of the present invention is exclusively for the prevention (suppression of liver function deterioration) for humans who have or are concerned about liver damage caused by such hepatitis virus, alcohol, stress, drugs or immune abnormalities. Or it can be used for the purpose of improvement (liver function improvement).

  Hereinafter, the present invention will be described in more detail by preparation examples and experimental examples. However, these experimental examples do not limit the present invention. In the following experimental examples, “%” means “% by weight” unless otherwise specified.

Preparation Example Production of globin proteolysate The details of the method for producing a globin proteolysate using bovine erythrocytes are shown below. After adding 250 liters of water to 100 kg of fresh bovine erythrocytes and sufficiently lysing, adjusting the pH to 2.8 by adding phosphoric acid, adding 2.6 × 10 ⁷ units of Aspergillus niger acid protease 30 The reaction was carried out at 3 ° C for 3 hours. After the reaction, the reaction solution was heated at 80 ° C for 30 minutes to stop the reaction, and then the pH was adjusted to 6.5 by adding an aqueous suspension of calcium hydroxide, 10 kg of diatomaceous earth was added, and a filter press was used. The resulting filtrate was spray-dried to obtain 23 kg of globin proteolysate powder. The molecular weight distribution of the obtained globin proteolysate was examined using gel filtration chromatography.

The chromatography was performed under the following conditions.
<Gel filtration chromatography>
Equipment: High-performance liquid chromatograph (Shimadzu Corporation, LC-6A type)
Column: PolyHYDROXYETHYL A, 5FLm, 9.4 × 200 m, PolyLCInc elution solvent: 50 mM formic acid flow rate: 0.5 ml / min Detection: UV absorption 221 nm.

  The gel filtration chromatogram of the globin proteolysate obtained by the gel filtration chromatography is shown in FIG.

Experimental Example 1 Galactosamine: Acute liver injury model The hepatitis caused by galactosamine is relatively similar to human viral hepatitis in symptoms and is often used as an acute hepatitis model.

  Six ddY mice were made into one group, and D-galactosamine was administered intraperitoneally at a rate of 600 mg / kg to induce liver damage in each mouse. The globin proteolysate was mixed in the feed at a rate of 0.1% or 1% and ingested for 7 days, and then D-galactosamine was administered. In addition, a group (normal group) in which only D-galactosamine and globin proteolysate were not administered and a group in which only D-galactosamine and water were administered (control group) were provided. 20 hours after administration of D-galactosamine, blood was collected from the abdominal vena cava, heparin was added, and plasma was separated. Using this collected blood, enzymes that are known to increase in blood concentration due to destruction of hepatocytes, ie, AST: aspartate aminotransferase (GOT) activity and ALT: alanine aminotransferase (GPT) activity Were measured with a commercially available measurement kit (manufactured by Wako Pure Chemical Industries, Ltd.). In addition, the liver was removed and the total amount of liver lipid was measured. And the increase / decrease of these enzyme activities and total liver lipid amount was made into the parameter | index of a liver disorder.

  The results of increase / decrease in enzyme activity (AST activity, ALT activity) and total liver lipid amount are shown in FIGS. As shown in FIGS. 2 and 3, in the control group to which D-galactosamine was administered, the activity values of both enzymes (AST, ALT) were remarkably increased as compared with the normal group, and necrosis of hepatocytes occurred. Was suggested. On the other hand, in the group administered with globin proteolysate (GD), the increase in both enzyme activities (AST activity and ALT activity) by D-galactosamine was greatly suppressed, and the globin proteolysate (GD) was excellent. It was shown to have an effect of improving liver damage. In addition, as shown in FIG. 4, the total liver lipid amount was significantly increased in the control group administered with D-galactosamine compared to the normal group, but this increase was suppressed by administration of GD. From now on, it was confirmed that globin proteolysate (GD) has an effect of improving liver damage. In addition, * mark in each figure has shown that there exists a statistically significant difference compared with a control group (p <0.05, t-test).

Experimental Example 2 Ethionine: Alcoholic Liver Damage Model Liver damage caused by ethionine is used as a model for alcoholic liver damage.

  Eight ddY mice were grouped and DL-ethionine was administered subcutaneously at a rate of 600 mg / kg to induce liver damage in each mouse. The globin proteolysate was mixed with the feed at a rate of 1% and ingested for 14 days, and then DL-ethionine was administered. In addition, a group in which only DL-ethionine and globin proteolysate were not administered and only water was administered (normal group), and a group in which only DL-ethionine and water were administered (control group) were provided.

  Blood was collected from the abdominal vena cava 20 hours after the administration of DL-ethionine, heparin was added, and plasma was separated. Using this collected blood, enzymes that are known to increase in blood concentration due to destruction of hepatocytes, ie, AST: aspartate aminotransferase (GOT) activity and ALT: alanine aminotransferase (GPT) activity Were measured with a commercially available measurement kit (manufactured by Wako Pure Chemical Industries, Ltd.). In addition, the liver was removed and the total amount of liver lipid was measured. And the increase / decrease in these enzyme activities and the total amount of liver lipids was used as an index of liver injury.

  The results of increase / decrease in enzyme activity (AST activity, ALT activity) and total liver lipid amount are shown in FIGS. 5, 6 and 7, respectively. As shown in FIGS. 5 and 6, in the control group to which DL-ethionine was administered, the activity values of both enzymes (AST, ALT) were significantly higher than those in the normal group, and necrosis of hepatocytes occurred. Was suggested. In contrast, in the group administered with globin proteolysate (GD), the increase in both enzyme activities (AST activity and ALT activity) due to DL-ethionine administration was greatly suppressed, which is superior to globin proteolysate (GD). It was shown to have an effect of improving liver damage. In addition, the total lipid level in the control group to which D-DL-ethionine was administered was significantly higher than that in the normal group, but this increase was suppressed by GD administration. Proteolysate (GD) was found to have an effect of improving liver damage. In addition, * mark in each figure has shown that there exists a statistically significant difference compared with a control group (p <0.05, t-test).

Example 3 In vitro test using primary cultured hepatocytes Using collagenase perfusion method, hepatocytes were isolated from rats (Wistar, male, 6-8 weeks old) and cultured overnight at 37 ° C. and 5% CO ₂ conditions. did. A medium containing 5 mM carbon tetrachloride (dissolved in DMSO: liver injury inducer) was added to the hepatocytes and incubated for 24 hours. The activity of lactate dehydrogenase (LDH), which is a marker of liver damage leaking into the medium, was measured using an LDH measurement kit (cytotoxicity detection kit: Roche Diagnostics).

The obtained result is shown in FIG. As can be seen from the figure, the LDH activity when DMSO alone was added to the medium was 0.1 unit / ml or less, but when carbon tetrachloride (CCl ₄ ) was dissolved in DMSO and added to the medium, LDH The activity increased to about 0.6 unit / ml, and hepatocellular injury was induced. On the other hand, when 1 mg / ml of globin proteolysate (GD) was added to the medium at the same time as carbon tetrachloride, the increase in LDH activity was remarkably suppressed, and was almost the same as when only DMSO was added. From these results, it was revealed that globin proteolysate (GD) has an effect of suppressing hepatocellular injury, that is, GD has an effect of preventing hepatocellular injury. In addition, ** mark in a figure has shown that there exists a statistically significant difference compared with the culture medium which added 5 mM carbon tetrachloride (p <0.01, t-test).

  As shown in the above experimental examples, in the liver injury model using mice, the globin proteolysate increases the AST and ALT values, which are indicators of liver injury, when administered before liver injury induction treatment. Suppressed and suppressed increase in total liver lipid content. In addition, cell damage induced by carbon tetrachloride was also significantly suppressed in rat primary hepatocytes. From these facts, the globin proteolysate of the present invention is useful as an agent for improving or preventing liver damage and can be used effectively for such purposes.

[Prescription example]
A composition having the following formulation was prepared using a globin proteolysate prepared according to the method described in the above Preparation Example.

Formulation Example 1 Soft Drinks Soft drinks were prepared according to the following formulation.

<Soft drink formula>
Reduced maltose starch syrup 5.0g
Erythritol 5.0g
Globin proteolysate 2.0g
Sour seasoning 0.2g
Preservative 0.01g
Perfume
Purified water
Total 100 ml.

  After dissolving 50 kg of reduced maltose starch syrup and 50 kg of erythritol in about 800 liters of purified water, add 20 kg of globin proteolysate, 2 kg of acidulant, and 0.1 kg of preservative in this order, and after complete dissolution, Purified water was added to make a total volume of 1000 liters, and a soft drink with liver damage improving action was prepared.

Formulation Example 2 Chocolate
To 100 g of chocolate, 1.5 g of the globin proteolysate prepared in Preparation Example was added to prepare a chocolate having an action for improving liver damage.

Formulation Example 3 Green Tea Beverage 8 kg of green tea was placed in 300 L of hot water at 80 ° C. and extracted at the same temperature for 4 minutes. The obtained extract was cooled and then centrifuged, and a clear supernatant was collected to obtain a green tea extract. To this extract, 0.4 kg of vitamin C was added, and 10 kg of the globin proteolysate prepared in Preparation Example was added, and the final volume was adjusted to 1000 L with hot water. This was heated to 85 ° C. or higher, filled into a metal can, and sterilized by retort (125 ° C., 5 minutes) to prepare a green tea beverage having an effect of improving liver damage.

Formulation Example 4 Chewing Gum Chewing gum was prepared according to the following formulation.

<Chewing gum formula>
Gum base 30 parts by weight Sugar 29 parts by weight Corn syrup 10 parts by weight Glycerin 1 part by weight
30 parts by weight of globin proteolysate
Total 100 parts by weight.

  First, gum base, sugar, corn syrup, and glycerin were mixed, the globin proteolysate prepared in Preparation Example was added, and the mixture was uniformly kneaded while keeping the temperature at 50 ° C. with a mixer. After cooling, it was pressure-molded with a roller to prepare a chewing gum having a plate-like liver injury-improving effect containing 300 mg of globin proteolysate as an active ingredient per sheet.

Formulation Example 5 Supplement The following ingredients are kneaded and granulated by a conventional method, dried and compressed into tablets, and about 65% by weight (200 mg) of globin proteolysate as an active ingredient in 1 tablet (310 mg) Tablets containing in proportion were obtained. The said tablet can be provided as a supplement which has the liver disorder improvement effect of a globin proteolysate.
<Prescription> per tablet
200 mg globin proteolysate
Lactose 100mg
Sucrose fatty acid ester 10mg
Total 310mg.

It is a gel chromatogram of a globin proteolysate. It is the graph which looked at the effect of the globin proteolysate (GD) with respect to galactosamine-induced liver injury using aspartate aminotransferase (GOT) [AST (GOT)] activity as an index (Experimental Example 1). It is the graph which looked at the effect of the globin proteolysate (GD) with respect to galactosamine-induced liver injury using alanine aminotransferase (GPT) [ALT (GPT)] activity as an index (Experimental Example 1). It is the graph which looked at the effect of the globin proteolysate (GD) with respect to the galactosamine-induced liver injury using the amount of total liver lipids as an index (Experimental example 1). It is the graph which looked at the effect of the globin proteolysate (GD) with respect to ethionine-induced liver damage using AST (GOT) as an index (Experimental Example 2). It is the graph which looked at the effect of the globin proteolysate (GD) with respect to ethionine-induced liver injury using ALT (GPT) as an index (Experimental Example 2). It is the graph which looked at the effect of the globin proteolysate (GD) with respect to ethionine-induced liver injury using the amount of total liver lipids as an index (Experimental Example 2). It is the graph which looked at the effect of the globin proteolysate (GD) with respect to hepatocyte damage induced by carbon tetrachloride using lactate dehydrogenase (LDH) as an index (Experimental Example 3).

Claims (3)

Hepatocyte disorder improving agent comprising as an active ingredient the globin proteolysate. Hepatocyte disorder improving agent according to claim 1 is hepatocellular disorder the hepatocellular damage is caused hepatitis virus, alcohol, stress, by drugs or immune disorder. Hepatocyte disorder improving agent according to claim 1 or 2 which is a prophylactic or therapeutic agent for alcoholic liver cell damage.

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