JP2008214265A - Interleukin-11 production promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substance promoting the production of IL-11 in a living organism (interleukin-11 production promoter) that is highly safe, can be continuously taken for long terms and exhibits excellent preservability. <P>SOLUTION: The interleukin-11 production promoter comprises lactoferrin as an effective ingredient. A food additive for promoting production of interleukin-11 comprises lactoferrin as an effective ingredient. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an interleukin-11 production promoter containing lactoferrin as an active ingredient. More specifically, the present invention relates to an interleukin-11 production promoter that is effective in protecting liver function, promoting platelet hematopoiesis, promoting differentiation of nerve cells, promoting osteoclast formation and improving bone metabolism through its function.

  The human interleukin-11 precursor consists of 199 amino acids, and 21 signal peptides are cleaved, and mature interleukin-11 consisting of 178 amino acids is secreted. Interleukin-11 (hereinafter sometimes abbreviated as IL-11) is expressed in various cells derived from the mesenchymal system, such as fibroblast, keratinocyte, chondrocyte, trophoblast, synoviocyte, bone marrow stroma cell, and osteoblast. Expressed, and expression is triggered by inflammatory cytokines and hormones.

  Interleukin-11 (IL-11) contains preadipocyte differentiation into adipocytes, increased acute phase protein production from hepatocytes, osteoclast production, neuronal differentiation, and spermatogenesis. There are various biological activities. In addition, IL-11 has various anti-inflammatory effects, including suppression of sepsis, suppression of LPL activity, suppression of apoptosis of vascular endothelial cells, protective action of lung mucosa / gastrointestinal mucosa, protective effect of liver damage, protective action of joint connective tissue degradation Etc. are known (Non-Patent Document 1).

  Prophylactic / therapeutic agents using such an interleukin-11 action on the immune system, anti-inflammatory action, and hematopoietic action have been disclosed (Patent Documents 1 to 4).

  Interleukin-11, which is a physiologically active protein, is produced by a recombinant gene manipulation method, and a formulation has been developed and is beginning to be provided to the medical field. However, as disclosed in Patent Document 5, IL-11 is relatively stable in a pH range near neutral in an aqueous solution, but it hydrolyzes depending on temperature. It was difficult to store for a long period of time, and in fact, it was a situation that had to be a freeze-dried preparation. In order to obtain a freeze-dried preparation, the IL-11 solution is rapidly cooled to −30 ° C. or lower to be in a supercooled state, frozen by breaking the supercooling of the solution, and then dried. However, a problem remains that white turbidity is observed temporarily during re-dissolution.

  A lyophilized preparation of a physiologically active protein is prepared by re-dissolving with water for injection in clinical use. At that time, it is necessary to quickly redissolve so that the presence or absence of abnormalities in the preparation, such as contamination of foreign substances, can be easily confirmed with the naked eye. For this reason, the freeze-dried preparation of IL-11 that causes white turbidity at the time of re-dissolution even temporarily is considered to be difficult to handle in clinical settings because it temporarily presents a confusing appearance such as contamination with foreign substances. It had been.

  Lactoferrin (hereinafter sometimes abbreviated as LF) is an iron-binding glycoprotein having a molecular weight of about 80 kilodaltons mainly contained in breast milk. E. coli, Candida, Clostridium, Staphylococci It is known as a milk protein having various actions such as antibacterial action against harmful microorganisms such as immunostimulatory action and antitumor action. Since lactoferrin is a milk-derived glycoprotein, it is highly safe and can be used for a long time. It itself is almost tasteless and odorless, and it is highly versatile as an additive for various foods, pharmaceuticals, and feeds. .

However, the effect of promoting production of interleukin-11 by lactoferrin has not been known.
JP-A-5-178756 JP-A-8-127539 JP-A-8-176008 Japanese Patent Application Laid-Open No. 8-176209 JP 2005-60377 A Biotherapy, Vol. 15, No. 6, 2001, pp. 697-709

  Therefore, in view of the problems of the IL-11 protein preparation as described above, the present inventors do not use a freeze-dried preparation of IL-11 protein but effectively induce IL-11 in vivo. Thus, the idea of avoiding these problems as a protein preparation by enjoying the physiological activity of IL-11 was obtained. That is, the object of the present invention is a substance that promotes IL-11 production in vivo (interleukin-11 production promoter), which is highly safe, can be used for a long period of time, and is preserved. The object is to provide a substance with excellent properties.

  Furthermore, the object of the present invention is effective in protecting liver function, promoting platelet hematopoiesis, promoting peripheral blood stem cell increase, promoting differentiation of nerve cells, promoting osteoclast production and improving bone metabolism through its function, etc. Another object of the present invention is to provide an interleukin-11 production promoter containing lactoferrin as an active ingredient, which is useful as a pharmaceutical for treatment and prevention.

  The inventors of the present invention have intensively conducted research for many years to search for interleukin-11 production promoters as described above. As a result, it has been found that lactoferrin, which is a milk-derived glycoprotein, has an effect of inducing IL-11, and the present invention has been completed.

Accordingly, the present invention is the following [1].
[1] An interleukin-11 production promoter containing lactoferrin as an active ingredient.

The present invention also includes the following [2] to [9].
[2] An interleukin-11 production promoter comprising lactoferrin as an active ingredient and a pharmaceutically acceptable carrier.
[3] A food and drink for promoting production of interleukin-11 containing lactoferrin as an active ingredient.
[4] The food or drink according to [3], wherein the food or drink is in the form of a health food, a functional food, a special-purpose food, a functional nutrition food, or a food for specified health use.
[5] Use of lactoferrin for producing an interleukin-11 production promoter (Use).
[6] Use of lactoferrin for producing food and drink for promoting production of interleukin-11 (Use).
[7] A food additive for promoting interleukin-11 production, comprising lactoferrin as an active ingredient.
[8] A feed for promoting production of interleukin-11, which contains lactoferrin as an active ingredient.
[9] A method of promoting interleukin-11 production in mammals by administering an interleukin-11 production promoter comprising lactoferrin as an active ingredient and a pharmaceutically acceptable carrier (Method).

  In addition, the interleukin-11 production promoter according to the present invention suitably exhibits the action of improving liver function, protecting liver function, maintaining bone density, increasing bone density, maintaining bone mass, or increasing bone mass. . Therefore, the present invention also resides in a liver function improving agent, liver function protecting agent, bone density maintaining agent, bone density increasing agent, bone mass maintaining agent, or bone mass increasing agent containing lactoferrin as an active ingredient. The present invention also includes a liver function improving agent, a liver function protecting agent, a bone density maintaining agent, a bone density increasing agent, and a bone mass maintaining agent, comprising lactoferrin as an active ingredient and a pharmaceutically acceptable carrier. Or a bone mass increasing agent. Furthermore, this invention exists also in the use (Use) of the lactoferrin for manufacturing these agents.

  Furthermore, this invention exists also in the food-drinks for improving liver function, liver function protection, bone density maintenance, bone density increase, bone mass maintenance, or bone mass increase which contain lactoferrin as an active ingredient. Moreover, this invention exists also in use (Use) of the lactoferrin for manufacturing these food-drinks.

  The present invention also includes a food additive containing lactoferrin as an active ingredient for improving liver function, protecting liver function, maintaining bone density, increasing bone density, maintaining bone mass, or increasing bone mass.

  The present invention also includes a feed for improving liver function, protecting liver function, maintaining bone density, increasing bone density, maintaining bone mass, or increasing bone mass, which contains lactoferrin as an active ingredient.

  The present invention also includes a liver function improving agent, a liver function protecting agent, a bone density maintaining agent, a bone density increasing agent, and a bone mass maintaining agent, comprising lactoferrin as an active ingredient and a pharmaceutically acceptable carrier. Alternatively, there is also a method (Method) for improving liver function, protecting liver function, maintaining bone density, increasing bone density, maintaining bone mass, or increasing bone mass by administering a bone mass increasing agent.

  The present invention provides a novel interleukin-11 production promoter. Since the interleukin-11 production promoter of the present invention can effectively induce interleukin-11 in vivo, liver function protection, platelet hematopoiesis promotion, peripheral blood stem cell increase promotion, nerve cell It is useful as a pharmaceutical for the purpose of promoting differentiation, promoting osteoclast production, and improving bone metabolism through its function.

  In addition, the interleukin-11 production promoter according to the present invention is highly safe, can be used for a long period of time, and is excellent in preservability, so that it can be easily handled in clinical settings.

  The interleukin-11 production promoter according to the present invention contains lactoferrin as an active ingredient. For this reason, since the interleukin-11 production promoter according to the present invention is derived from milk, it is highly safe for humans and animals, and is continuously used for a long time without worrying about side effects. Can be ingested. Furthermore, since lactoferrin, which is an active ingredient of the present invention, has heat resistance, is water-soluble, and is stable even in an aqueous solution, the interleukin-11 production promoter according to the present invention is stable as a drug or food and drink. is there. Furthermore, since the interleukin-11 production promoter according to the present invention can be administered orally, it is simple and highly versatile. Furthermore, the interleukin-11 production promoter according to the present invention can be stably produced in large quantities from raw materials such as milk or whey that are relatively inexpensive as biomaterials. Furthermore, since lactoferrin, which is an active ingredient of the present invention, is originally tasteless and odorless, it can be taken orally together with luxury products.

  Preferred embodiments of the present invention are described in detail below. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention. In the present specification, percentages are expressed by mass unless otherwise specified.

  The present invention is an interleukin-11 production promoter containing lactoferrin as an active ingredient.

  The interleukin-11 production promoter of the present invention is used for liver function protection, platelet hematopoiesis promotion, peripheral blood stem cell increase promotion, nerve cell differentiation promotion, osteoclast production promotion and improvement of bone metabolism through its function, etc. Since it exhibits an excellent effect, it can be applied as a pharmaceutical product that expects the medicinal effect.

  The lactoferrin used in the present invention is commercially available lactoferrin, mammals (eg, humans, cows, goats, sheep, horses, etc.) colostrum, transitional milk, regular milk, end milk, or processed products of these milks. Non-fat milk, whey, etc. as raw materials can be used, and for example, lactoferrin obtained by separation from the raw materials by a conventional method such as ion exchange chromatography can be used. Among them, it is preferable to use commercially available lactoferrin (for example, manufactured by Morinaga Milk Industry Co., Ltd.) manufactured on an industrial scale. Furthermore, it is also possible to use lactoferrin produced in microorganisms, animal cells, transgenic animals, etc. by genetic engineering techniques.

  The lactoferrin used in the present invention is a bovine, sheep, goat whose milk is traditionally used for food and drink among mammals when used in pharmaceutical compositions and foods and drinks for humans. Lactoferrin derived from milk such as is preferable, and milk lactate is particularly preferable. This is because these have been used for human consumption during historical years, and thus have a very high level of safety for humans. In addition, whey derived from milk is particularly suitable as a raw material for lactoferrin used in the present invention because it has the advantage that it can be obtained stably and in large quantities as a by-product of dairy production.

  Here, an example of a method for preparing lactoferrin (separation and purification of lactoferrin from raw materials such as milk) is shown below. First, an ion exchanger (for example, CM-Sepharose FF (trade name, manufactured by Amersham Pharmacia)) is packed in a column, passed through hydrochloric acid, and washed with water to equilibrate the ion exchanger. Subsequently, skim milk of pH 6.9 cooled to 4 ° C. is passed through the column, the permeate is collected, and again passed through the column in the same manner. Next, distilled water is passed through the column, and saline is passed through to obtain an eluate of the basic protein adsorbed on the ion exchanger. To this eluate, 80% saturation ammonium sulfate is added to precipitate the protein, and centrifuged to collect the precipitate. The collected precipitate is washed with an ammonium sulfate solution having a saturation degree of 80%, deionized water is added to dissolve the precipitate, and an ultrafiltration membrane module (for example, SLP0053 (trade name, manufactured by Asahi Kasei Co., Ltd.)) is obtained. Used to desalinate and freeze-dry to obtain powdered bovine lactoferrin. In this way, bovine lactoferrin having a purity of 95% by mass or more is obtained. The purity of lactoferrin in the present specification is a value obtained by measurement by liquid chromatography.

  In the effect of the interleukin-11 production promoter of the present invention, the metal content in lactoferrin is not particularly limited. In the present invention, apo-type lactoferrin obtained by removing iron from lactoferrin with hydrochloric acid, citric acid or the like; Metal saturated lactoferrin in a state of 100% saturation obtained by chelating with a metal such as iron, copper, zinc, manganese, etc .; and metal partially saturated lactoferrin in a state in which the metal is bound at various saturations of less than 100% Any one or a mixture of two or more selected from the group consisting of:

  As a preferred form of lactoferrin used in the present invention, human-derived lactoferrin or bovine-derived lactoferrin is exemplified, and one kind can be used alone, or a plurality of kinds can be used in combination.

  In these proteins, naturally there are mutations such as substitution, deletion, insertion, addition or inversion of one or more bases at one or more positions depending on the species, genus, individual, etc. There may also be mutations in the amino acids of the proteins encoded by genes with various mutations. The lactoferrin that can be used in the present invention includes those containing such mutations as long as the interleukin-11 production promoting activity is not impaired.

  In the present invention, the interleukin-11 production promoting effect based on lactoferrin can be measured by measuring the amount of interleukin-11 produced by molecular biological or immunological evaluation methods such as Western blotting, Northern blotting, or ELISA. it can. In addition, about this measuring method, it describes in detail in the test example or Example of this invention.

  The interleukin-11 production promoter of the present invention can be administered orally or parenterally to mammals including humans in combination with lactoferrin or a pharmaceutically acceptable pharmaceutical carrier. The dosage unit form of the pharmaceutical preparation of the present invention is not particularly limited and can be appropriately selected depending on the purpose of treatment. Specifically, tablets, pills, powders, solutions, suspensions, emulsions, granules, fine granules And capsules, syrups, elixirs, suppositories, injections, ointments, patches, eye drops, nasal drops and the like. In formulation, excipients, binders, disintegrants, lubricants, stabilizers, fluidity promoters, flavoring agents, flavoring agents, coloring agents, fragrances, diluents, which are commonly used for ordinary drugs as pharmaceutical carriers. Additives such as surfactants and solvents for injections can be used. Using these, a preparation of an interleukin-11 production promoter can be produced according to a conventional method.

  Examples of the binder include starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol and the like.

  Examples of the disintegrant include starch, hydroxypropyl starch, carboxymethylcellulose sodium, carboxymethylcellulose calcium, carboxymethylcellulose, and low-substituted hydroxypropylcellulose.

  Examples of the surfactant include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80 and the like.

  Examples of the lubricant include talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol and the like.

  Examples of the fluidity promoter include light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate and the like.

  The amount of lactoferrin contained in the preparation of the present invention is not particularly limited and may be appropriately selected. For example, all are usually 0.0001 to 75% by mass, preferably 0.001 to 50% by mass in the preparation. Is good.

  The administration method of the preparation of the present invention is not particularly limited, and is determined according to various preparation forms, the patient's age, sex, other conditions, the degree of symptoms of the patient, and the like. The dosage of the active ingredient in the preparation of the present invention is appropriately selected depending on the usage, patient age, sex, disease severity, other conditions, and the like. Usually, the amount of lactoferrin as an active ingredient is 0.01 to 2000 mg / kg / day, preferably 0.1 to 1500 mg / kg / day, particularly preferably 1 to 1000 mg / kg / day. It can be administered once or a plurality of times a day.

  The interleukin-11 production promoter of the present invention exhibits remarkable effects on liver function protection, platelet hematopoiesis promotion, peripheral blood stem cell increase promotion, nerve cell differentiation promotion, osteoclast production promotion, etc. It is useful as a medicament for treatment / prevention of diseases involving interleukin-11 such as thrombocytopenia, Crohn's disease, rheumatoid arthritis, hepatitis, osteoporosis and the like. Although the interleukin-11 production promoter of the present invention may be used alone, it can also be used in combination with known prophylactic / therapeutic agents for the diseases. By using in combination, the preventive / therapeutic effect of the disease can be enhanced. The prophylactic / therapeutic agent for the above-mentioned diseases to be used in combination may be contained as an active ingredient in the composition of the present invention, or it is not contained in the composition of the present invention and is combined as a separate drug to produce a product. You may combine at the time of use.

  Moreover, the interleukin-11 production promoter of this invention can add the lactoferrin which is an active ingredient as an additive with respect to a foodstuff or a drink, and can manufacture it in the form of the food-drinks composition, orally. Ingestion is possible.

  Examples of the form of such a food / beverage composition (food / drink for promoting production of interleukin-11) include beverages such as soft drinks, carbonated drinks, nutrient drinks, fruit juice drinks, and lactic acid bacteria drinks (concentrated concentrates of these drinks and Ice cream, ice sherbet, shaved ice, etc .; noodles such as buckwheat, udon, harsame, gyoza skin, cucumber skin, Chinese noodles, instant noodles; strawberry, chewing gum, candy, gum, chocolate , Tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, baked confectionery, etc .; fishery and livestock processed foods such as kamaboko, ham, sausage; dairy products such as processed milk, milk drinks, fermented milk, butter; , Breads; enteral nutritional foods, liquid foods, milk for childcare, sports drinks; functional foods such as foods for specified health use and health supplements

  The form of the functional food is preferably a granular, tablet or liquid supplement from the viewpoint of easily grasping the intake amount of the active ingredient.

  In the food / beverage product composition of the present invention, the amount of lactoferrin added is appropriately set according to the form of the food / beverage product composition, but is 0.0001 to 75% by mass, preferably 0.001 to 50% in a normal food or beverage. What is necessary is just to add so that it may become mass%.

  In the food and drink for promoting production of interleukin-11 according to the present invention, in addition to the lactoferrin which is an active ingredient, for example, sugar absorption-inhibiting saccharides such as lactulose, maltitol, and lactitol, and other saccharides Such as dextrin and starch; proteins such as gelatin, soybean protein and corn protein; amino acids such as alanine, glutamine and isoleucine; polysaccharides such as cellulose and gum arabic; fats and oils such as soybean oil and medium chain fatty acid triglyceride It can be included.

  The food / beverage products for promoting interleukin-11 production of the present invention are food / beverage products for which the use such as interleukin-11 production promotion is indicated, for example, “lactoferrin effective for promoting interleukin-11 production” is effective. It is preferable to sell as “a food or drink containing as a component”.

  The “display” act (display act) includes all acts for informing consumers of the use, and if it is a display that can recall and analogize the use, the purpose of the display Regardless of the contents of the display, the object / medium to be displayed, etc., all fall under the “display” act of the present invention. However, it is preferable to display in such an expression that the consumer can directly recognize the application. Specifically, the act of describing the above-mentioned use in the product or the product packaging relating to the food or drink of the present invention can be cited as a display act, and further, the product or product packaging describing the above-mentioned use is assigned, Display for distribution, transfer or delivery, importing, displaying advertisements on products, price lists or transaction documents for display or distribution, or listing the use for information Thus, an act provided by an electromagnetic (Internet etc.) method can be exemplified.

  On the other hand, the displayed content (display content) is a display approved by the government or the like (for example, a display that is approved based on various systems determined by the government and is performed in a mode based on such approval). It is preferable to attach such display contents to advertising materials at sales sites such as packaging, containers, catalogs, pamphlets, POPs, and other documents.

  Further, for example, indications as health food, functional food, enteral nutrition food, special purpose food, health functional food, food for specified health, functional nutrition food, quasi drug, etc. can be exemplified. In particular, a display approved by the Ministry of Health, Labor and Welfare, for example, a display approved by a food system for specific health use or a system similar thereto can be exemplified. Examples of the latter can include a display as a food for specified health use, a display as a condition specific food for specified health use, a display that affects the structure and function of the body, a display for reducing disease risk, etc. Speaking of the health promotion law enforcement regulations (April 30, 2003 Ministry of Health, Labor and Welfare Ordinance No. 86) labeling as food for specified health use (especially labeling the use of health), and similar The display can be listed as a typical example.

  The wording used to display the above uses is not limited to the wording “promotion of interleukin-11 production”, and the above interleukin-11 production promotion is possible even in other words. It is needless to say that the wording that expresses the effect is included in the scope of the present invention.

  The food additive for promoting production of interleukin-11 of the present invention can be produced by including lactoferrin when producing various food additives. As lactoferrin, the same thing as the lactoferrin used for manufacture of the interleukin-11 production promoter etc. which were mentioned above can be used.

  In addition to lactoferrin as an active ingredient, food additives can contain additives such as commonly used excipients as desired. Further, other components known as food additives can be included as desired. There is no restriction | limiting in particular in the form of a food additive, It can take the usual form of a food additive, such as a powder, a granule, a tablet, a liquid.

  The food additive for promoting production of interleukin-11 of the present invention can be added to produce a food or drink for promoting production of interleukin-11. Therefore, if it contains lactoferrin as an active ingredient and can be added to produce a food or drink for promoting interleukin-11 production, the food for promoting interleukin-11 production of the present invention It is contained in the range of additives.

  About the act of the display regarding a food additive, it is as having mentioned above about food-drinks. The wording used to display the above uses is not limited to the wording “for promoting interleukin-11 production”, and the above-mentioned interleukin-11 production is possible even in other words. Needless to say, any word expressing the effect of promotion is included in the scope of the present invention.

  Furthermore, the interleukin-11 production promoter of the present invention can be produced in the form of a feed composition by adding lactoferrin to the feed, and is orally administered to general mammals, livestock, fish farms and pets. Can be administered. Examples of such a feed composition (feed for promoting production of interleukin-11) include pet food, livestock feed, and fish feed. There is no particular limitation on the form of the feed composition, and it can be a normal form of each feed, but it is easy to grasp the intake amount of the active ingredient if it is a granular, tablet or liquid supplement This is preferable.

  In addition to the active ingredient according to the present invention, the feed of the present invention includes, for example, cereals (for example, corn, wheat, barley, rye, milo, etc.); moss (for example, soybean oil meal, rapeseed oil meal, coconut oil meal, flaxseed) Vegetable oils such as oil cakes, and production rice cakes such as corn gluten meal and corn jam meal); moss (eg, bran, wheat straw, rice bran, defatted rice meal, etc.); animal feeds (eg fish meal, defatted) Powdered milk, whey, yellow grease, tallow, etc.); yeasts (eg, Tral yeast, beer yeast, etc.); mineral feeds (eg, tricalcium phosphate, calcium carbonate, etc.); fats and oils; simple amino acids; Can be produced as a feed composition.

  In the feed composition of the present invention, the amount of lactoferrin added is appropriately set according to the form of the feed composition, but is 0.0001 to 75% by mass, preferably 0.001 to 50% by mass in normal feed. It may be added as follows.

[Example]
EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

  150 g of bovine lactoferrin (Morinaga Milk Industry Co., Ltd.), lactulose powder (Morinaga Milk Industry Co., Ltd.) 100 g, maltextrin (Matsuya Chemical Industry Co., Ltd.) 635 g, skim milk powder (Morinaga Milk Industry Co., Ltd.) 85 g, Stevia sweetener (San-Eigen F Each powder of 1 g of F eye), 5 g of yogurt flavor (manufactured by Saneigen F.F.I.), and 24 g of glycerin fatty acid ester preparation (manufactured by Riken Vitamin Co., Ltd.) was added and mixed uniformly. (Produced by Hata Steel Co., Ltd.), 0.5 g per tablet, and the above mixed powder is continuously tableted at a tableting speed of 12 tablets / minute and a pressure of 9.8 KPa. Interleukin-11 1800 tablets (about 900 g) containing lactoferrin, a drug that has a production promoting effect and is useful for increasing peripheral blood stem cells and promoting differentiation of nerve cells, were produced.

  An aqueous phase was prepared in a tank by dissolving 10.8 kg of whey protein hydrolyzate (manufactured by Morinaga Milk Industry Co., Ltd.), 36 kg of dextrin (manufactured by Showa Sangyo Co., Ltd.), and a small amount of water-soluble vitamins and minerals in 200 kg of water. Separately, soybean salad oil (manufactured by Taiyo Yushi Co., Ltd.) 3 kg, palm oil (manufactured by Taiyo Yushi Co., Ltd.) 8.5 kg, safflower oil (manufactured by Taiyo Yufu Co., Ltd.) 2.5 kg, lecithin (manufactured by Ajinomoto Co., Inc.) 0.2 kg, An oil phase was prepared by mixing and dissolving 0.2 kg of a fatty acid monoglyceride (manufactured by Kao Corporation) and a small amount of a fat-soluble vitamin. The oil phase was added to the aqueous phase in the tank, stirred and mixed, then heated to 70 ° C., and further homogenized with a homogenizer at a pressure of 14.7 MPa. Next, the mixture was sterilized at 90 ° C. for 10 minutes, concentrated and spray-dried to prepare about 59 kg of intermediate product powder. To this intermediate product powder 50 kg, sucrose (Hokuren Co., Ltd.) 6.8 kg, amino acid mixed powder (Ajinomoto Co., Inc.) 167 g, and bovine lactoferrin (Morinaga Milk Industry Co., Ltd.) 60 g are added and mixed uniformly. About 56 kg of enteral nutritional meal powder containing lactoferrin, which has a production promoting effect on -11 and is useful for protecting liver function, was produced.

[Test example]
Next, the present invention will be described in detail with reference to test examples.

[Test Example 1]
This study was conducted to examine the effect of promoting IL-11 production in the liver by oral administration of lactoferrin (hereinafter sometimes referred to as LF) and the effect of improving the liver damage caused by it in a drug-induced liver injury model. It was.

(1) Test method Male 6-week-old ICR mice (Nippon Charles River) were preliminarily raised for 1 week. D-galactosamine hydrochloride (Wako Pure Chemical Industries, Ltd.) was dissolved in physiological saline to prepare a 50 mg / ml solution, and a solution corresponding to 500 mg / kg body weight was administered intraperitoneally. For 15 days from the day of administration of the D-galactosamine solution, BSA (bovine serum albumin: manufactured by Sigma) and lactoferrin (abbreviated as LF; manufactured by Morinaga Milk Industry Co., Ltd.) were dissolved in distilled water to prepare a 30 mg / ml solution. A volume of 300 mg / kg body weight was administered intragastrically with an oral sonde. In addition, the group which did not perform intraperitoneal administration and intragastric administration was made into the non-administration group. On the 14th day after administration of the first galactosamine solution, the same amount of galactosamine solution was intraperitoneally administered again (second administration).

  Separately, in order to examine the minimum effective dose of lactoferrin, LF: 3 mg / ml, 10 mg / ml, and 30 mg / ml solutions were prepared, and 30 mg / kg body weight and 100 mg / kg body weight were obtained according to the above methods, respectively. The effect was compared with a volume of 300 mg / kg body weight.

  Twenty-four hours after the final oral administration of BSA and lactoferrin, heart blood and liver were collected under chloroform anesthesia.

  RNA extraction from the liver was performed using RNA-Bee (catalog number: CS-105B, manufactured by Tel-Test Corp.), and purification of mRNA from the extracted RNA was Turbo-DNA Free (catalog number: 1907, Ambion). Made). CDNA was prepared from the obtained mRNA using ExScript RT reagent kit (catalog number: RR035A, manufactured by Takara Shuzo), SYBR Premix Ex Taq (catalog number: RR041A, manufactured by Takara Shuzo), and mouse interleukin-11 primer. Was used for real-time PCR on mouse interleukin-11, and the mRNA expression level of mouse IL-11 was quantified. As a standard sample, the amount of mouse GAPDH mRNA expression was quantified, and this value divided by the value of mouse IL-11 mRNA expression was used as a relative expression for comparison. The average value of the non-administered group was 1, and the expression level of the BSA and lactoferrin administered group was expressed.

  Protein extraction from the liver was performed using a Mammalian cell lysis kit (catalog number: MCL-1; manufactured by Sigma). Protein quantification was performed using BCA Protein Assay Reagent (catalog number: # 23227, manufactured by Pierce). 50 mg of the prepared sample was electrophoresed using NuPAGE 4-12% Bis-Tris Gel (catalog number: NP0321BOX, manufactured by Invitrogen) and transferred to a membrane membrane for protein transfer Hybond-P (catalog number: RPN2020F, manufactured by Amersham). After that, immunoblotting was performed using an anti-mouse IL-11 antibody (catalog number: AF-418-NA, manufactured by R & D systems), and ECL Plus (catalog number: RPN2132, manufactured by Amersham) and Hyperfilm ECL (catalog number). : RPN2103K (manufactured by Amersham) was used to detect mouse IL-11. IL-11 detected on the film was quantified using a densitometer (model number: GS-700, manufactured by Bio-Rad) and analysis software Quantity One (Ver. 4.41, manufactured by Bio-Rad).

  Serum is collected from a heart blood sample, and GOT (glutamate oxaloacetate transaminase) and GPT (glutamate pyruvate transaminase) concentrations are measured using transaminase CII test Wako (catalog number: 431-30901, manufactured by Wako Pure Chemical Industries, Ltd.). did.

(2) Test results The results of this test are shown in Figs.

  FIG. 1 shows the expression level of IL-11 mRNA in the liver as a relative expression level with respect to the non-administered group. As a result, the relative expression level of mRNA in each group was 1.00 ± 0.94 in the non-administration group and 0.40 ± 0.28 in the BSA administration group, whereas the LF (lactoferrin) administration group Then, it was 1.41 ± 2.74 (the result of the t test was that the significant difference from the BSA group was p = 0.0585). From this, it was confirmed that the mRNA expression level of IL-11 was increased by the administration of lactoferrin.

  FIG. 2 shows the expression level of IL-11 protein in the liver (ng / 50 μg protein in liver tissue). As a result, the expression level of IL-11 protein in each group was 0.55 ± 0.52 (ng / 50 μg) in the non-administered group (the group expressed as non-treat in FIG. 2), and in the BSA-administered group, 0.99 ± 1.00 (ng / 50 μg), whereas in the LF administration group, it was 1.66 ± 1.02 (ng / 50 μg). Thus, it was confirmed that the expression level of IL-11 protein was significantly increased. From this, it was confirmed that the expression level of IL-11 protein increases by administration of lactoferrin.

  Furthermore, FIG. 3 has shown the inhibitory effect with respect to D-galactosamine induction liver damage by lactoferrin oral administration. The left diagram of FIG. 3 (serum GOT) shows the inhibitory effect on the D-galactosamine-induced liver injury using the amount of serum GOT (U / ml) as an index. As a result, the GOT amount in each group was 77.97 ± 13.83 in the non-administered group, whereas BSA administration after the second galactosamine administration (GalN × 2 in the left diagram of FIG. 3). 164.22 ± 105.55 in the group and 78.41 ± 27.63 in the LF administration group. As a result of the t-test, there is a significant difference between the BSA administration group and the LF administration group after the second galactosamine administration. confirmed. From this, it was confirmed that liver function was improved (liver function protection) by administration of lactoferrin (LF) having an IL-11 production promoting action.

  Moreover, the right figure (serum GPT) of FIG. 3 has shown the inhibitory effect which used the amount of GPT in serum (U / ml) with respect to D-galactosamine-induced liver injury as an index. As a result, the amount of GPT in each group was 65.10 ± 24.94 in the non-administered group, whereas 248.62 ± 209.92 in the BSA-administered group after the second administration of galactosamine, In the LF administration group, it was 73.18 ± 30.35, and as a result of the t test, a significant difference was confirmed between the BSA administration group and the LF administration group after the second administration of galactosamine. From this, it was confirmed that liver function was improved (liver function protection) by administration of lactoferrin (LF) having an IL-11 production promoting action.

  Further, FIG. 4 shows the dose-dependent liver injury-suppressing effect of lactoferrin by comparing the blood GOT concentration (the upper diagram in FIG. 4) and the blood GPT concentration (the lower diagram in FIG. 4). As a result, the blood GOT concentration (unit / ml) was 61.3 ± 5.0 in the non-administration group, whereas it was 112.8 ± 35.5 in the BSA administration group, and 30 mg / ml in the LF administration group. In the kg body weight administration group (LF30), 89.5 ± 21.5, in the 100 mg / kg body weight administration group (LF100), 69.4 ± 9.6, in the 300 mg / kg body weight administration group (LF300), 54. As a result of t-test, a significant difference was confirmed between the BSA group and the administration group (LF100 and LF300) having an LF of 100 mg / kg body weight or more.

  Furthermore, the blood GPT concentration (unit / ml) was 34.9 ± 14.2 in the non-administered group, compared with 61.3 ± 32.4 in the BSA-administered group, 30 mg / kg body weight in the LF-administered group. The administration group (LF30) was 53.8 ± 35.1, the 100 mg / kg body weight administration group (LF100) was 34.2 ± 8.5, and the 300 mg / kg body weight administration group (LF300) was 25.8 ± 5. As a result of t-test, a significant difference was confirmed between the BSA group and the administration group (LF300) having an LF of 300 mg / kg body weight.

  From the above experiment, lactoferrin has an effect as an interleukin-11 production promoter, and further, this action as an interleukin-11 production promoter has effects of liver function protection and liver function improvement. I understood it.

[Test Example 2]
This test was conducted to examine the effect of promoting IL-11 production in femur bone marrow and small intestinal epithelial tissue by oral administration of lactoferrin and the bone density increasing effect resulting therefrom in an aging-promoting mouse model (SAMP6).

(1) Test method SAMP6 mouse (purchased from Japan SLC), which is used for research as an osteoporosis model (senile osteoporosis model) in which aging is promoted by a genetic disease and bone density decrease starts from a young age in particular Breeding started at the age of 6 weeks and was raised to 5 months of age, and then the mice were subjected to experiments.

  Bovine serum albumin (BSA: Sigma) was dissolved in physiological saline to adjust the concentration to 30 mg / ml and used as a control sample. In addition, lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) was dissolved in physiological saline and the concentrations were adjusted to 10 mg / ml, 30 mg / ml, and 100 mg / ml, respectively, and used as test samples.

  BSA administration group: 300 mg / kg body weight, LF administration group: 100 mg / kg body weight, 300 mg / kg body weight, 1000 mg / kg body weight so that each sample was given to SAMP6 mice by oral sonde in the stomach for 4 weeks every day The femur and small intestine epithelial tissue were collected under chloroform anesthesia 3 days after the final administration (after completion of administration).

  Extraction of RNA from femur bone marrow and small intestine epithelial tissue and measurement of IL-11 by real-time PCR were performed in the same manner as in Test Example 1 described above. Further, the remaining one of the collected femurs was fixed with ethanol, and the bone density was measured using animal micro CT Latheta (manufactured by Aloka).

(2) Test result The result of this test is shown in FIGS. FIG. 5 shows the expression level of IL-11 mRNA in the femur of SAMP6 mice. FIG. 6 shows the expression level of IL-11 mRNA in the small intestinal epithelium of SAMP6 mice. Furthermore, FIG. 7 shows the femur plane bone density of SAMP6 mice. In addition, the measured value in FIG. 5, FIG. 6 was shown by the relative expression level which set the average of the measured value in a BSA administration group to 1.

  As a result, as shown in FIG. 5, the mRNA expression level of IL-11 in the femur was 24.10 ± 31.24 in the LF 100 mg / kg body weight administration group compared to BSA (1.00 ± 0.38). The LF 300 mg / kg body weight administration group was 21.80 ± 16.95, the LF 1000 mg / kg body weight administration group was 9.62 ± 13.82, and as a result of the t-test, the BSA administration group and the LF 300 mg / kg body weight administration group The IL-11 mRNA expression level was significantly increased. From this, it was confirmed that the IL-11 mRNA expression level in the femur increases by the administration of lactoferrin.

  In addition, as shown in FIG. 6, the IL-11 mRNA expression level in the small intestinal epithelium was 1.11 ± 0.49 in the LF 100 mg / kg body weight administration group compared to BSA (1.00 ± 0.59). The LF 300 mg / kg body weight administration group was 2.32 ± 1.87, the LF 1000 mg / kg body weight administration group was 1.35 ± 0.84, and as a result of the t-test, between the BSA administration group and the LF 300 mg / kg body weight administration group, Significant increase in IL-11 mRNA expression level was confirmed. From this, it was confirmed that the expression level of IL-11 mRNA in the small intestinal epithelium increases by the administration of lactoferrin.

Furthermore, as shown in FIG. 7, the SAMP6 mouse planar bone density (mg / cm ² ) was 73.5 ± 4.3 in the BSA administration group, while 85.6 ± 6. 6 in the LF 100 mg / kg body weight administration group. 2, 81.7 ± 5.9 in the LF 300 mg / kg body weight administration group, and 82.7 ± 6.4 in the LF 1000 mg / kg body weight administration group. As a result of the t-test, between the BSA administration group and any LF administration group In addition, a significant increase in planar bone density was confirmed. From this, it was confirmed that the bone density of a mouse | mouth increases by administration of the lactoferrin (LF) which has an IL-11 production promotion effect.

  From the above experiment, lactoferrin has an action as an interleukin-11 production promoter, and further, the action as this interleukin-11 production promoter is to maintain bone density and increase bone density (maintenance of bone mass and bone It was found that the effect of (mass increase) was brought about.

FIG. 1 is a view showing the expression level of liver IL-11 mRNA in a GalN hepatitis induction model mouse. FIG. 2 is a view showing the expression level of liver IL-11 protein in GalN hepatitis-induced model mice. FIG. 3 is a diagram showing the liver injury-suppressing effect of hepatitis-induced model mice. FIG. 4 is a diagram showing the dose-dependent liver injury-suppressing effect of hepatitis-inducing model mice. FIG. 5 is a graph showing IL-11 mRNA expression level in bones of senile osteoporosis model mice. FIG. 6 is a graph showing the expression level of IL-11 mRNA in the intestinal epithelial tissue of senile osteoporosis model mice. FIG. 7 is a diagram showing the planar bone density of a senile osteoporosis model mouse.

Claims (2)

  An interleukin-11 production promoter containing lactoferrin as an active ingredient.   A food additive for promoting production of interleukin-11, which contains lactoferrin as an active ingredient.

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