JP7177440B2 - Composition containing organic selenium compound - Google Patents

Description

The present invention relates to a composition containing an organic selenium compound as an active ingredient. More specifically, the present invention reduces oxidative stress and prevents ischemic diseases, improves fatty liver, improves glucose intolerance, improves metabolic syndrome including dyslipidemia, improves protection of disorders associated with metabolic syndrome, suppresses carcinogenesis, and prevents systemic diseases. The present invention relates to a composition, a food, a food composition, and a pharmaceutical composition for suppressing decreased immunocompetence, suppressing arteriosclerosis, preventing dementia and improving brain function, protecting corneal epithelial cells and improving dry eye, and/or suppressing inflammation.

It has been found that selenoneine, which is an organic selenium compound, has a strong antioxidant ability (radical scavenging activity) and exhibits a bioantioxidant effect on human cells (Patent Document 1). Antioxidant substances remove reactive oxygen species in the body and exhibit various physiological activities. Since selenium is an essential trace element for the human body, ingestion of selenoneine, which is organic selenium derived from seafood, can compensate for insufficient selenium intake from the diet. Therefore, application to functional foods containing selenoneine as an active ingredient is expected.

Since eating fish is known to have preventive effects against lifestyle-related diseases such as cancer and diabetes, as well as anti-aging effects, selenoneine is thought to contribute to the prevention of such lifestyle-related diseases and anti-aging. However, the preventive effect of selenoneine on lifestyle-related diseases, its molecular mechanism, and interactions between selenoneine and other biomolecules have not been verified. In particular, the effect of improving antioxidant capacity such as radical scavenging activity by selenoneine incorporated into human or animal cells and enhancing health functionality has not been investigated so far. Therefore, it was necessary to verify the bioactivity of selenoneine against biomolecules, model cells, and living organisms, and to verify the wide-ranging effectiveness of selenoneine for the development and practical application of new uses.

Japanese Patent No. 5669056

The present invention uses mammalian cells and rodents to analyze the effectiveness of selenoneine, an organic selenium compound, on the health and function of the body. The purpose is to demonstrate the effect of improving oxidative stress disorders that occur in vivo, such as disease prevention, dry eye improvement, and inflammation suppression, and reducing stress. Thereby, a selenium compound-containing composition containing selenoneine having novel food functionality such as anti-metabolic syndrome effect is provided.

Oxidative stress is defined as an unfavorable state for the body due to the imbalance between the oxidative reaction and the antioxidant reaction of the body caused by active oxygen, active nitrogen species, and free radicals. and oxidative stress are involved in a wide variety of diseases such as inflammation, arteriosclerosis, cancer, aging, cranial nerve disease, respiratory disease, cataract, skin disease, digestive disease, heart disease, and hypertension. It is known to play a central role in pathogenesis. Selenoneine, which has a strong radical scavenging activity, reduces active oxygen, free radicals, and oxidative stress generated in the body.By verifying the preventive effect of diseases caused by these oxidative stress, selenoneine is used as an active ingredient. New foods can be developed.

The present inventors have biochemically analyzed the health functionality of selenoneine and found that ingestion of a selenium compound-containing composition has the effect of reducing stress to protect against brain damage caused by cerebral ischemia and preventing stress-induced disorders. The present inventors have found that the present invention has been completed.

That is, the present invention is as follows.
[1] Formula I:

[In the formula,
R is hydrogen, ergothionyl, glutathionyl, or cysteinyl]
Oxidative stress reduction, oxidized lipid suppression, ischemic disease prevention, metabolic syndrome improvement, glucose intolerance improvement, improvement of protection and improvement of disorders associated with metabolic syndrome including lipid metabolism abnormality, containing an organic selenium compound represented by as an active ingredient, A composition for suppressing carcinogenesis, suppressing deterioration of systemic immune function, suppressing vascular endothelial damage, suppressing arteriosclerosis, preventing dementia, improving brain function, protecting corneal epithelial cells, improving dry eye, or suppressing inflammation.

[2] The organic selenium compound is represented by formula II:

The composition according to [1] above, which is 3-(2-hydroseleno-1H-imidazol-5-yl)-2-(trimethylammonio)propanoate represented by.

[3] Formula III:

Oxidative stress reduction, oxidized lipid suppression, ischemic disease prevention, metabolic syndrome improvement, glucose intolerance improvement, improvement of protection and improvement of disorders associated with metabolic syndrome including lipid metabolism abnormality, containing an organic selenium compound represented by as an active ingredient, A composition for suppressing carcinogenesis, suppressing deterioration of systemic immune function, suppressing vascular endothelial damage, suppressing arteriosclerosis, preventing dementia, improving brain function, protecting corneal epithelial cells, improving dry eye, or suppressing inflammation.

[4] Formula IV:

Oxidative stress reduction, oxidized lipid suppression, ischemic disease prevention, metabolic syndrome improvement, glucose intolerance improvement, improvement of protection and improvement of disorders associated with metabolic syndrome including lipid metabolism abnormality, containing an organic selenium compound represented by as an active ingredient, A composition for suppressing carcinogenesis, suppressing deterioration of systemic immune function, suppressing vascular endothelial damage, suppressing arteriosclerosis, preventing dementia, improving brain function, protecting corneal epithelial cells, improving dry eye, or suppressing inflammation.

[5] Metabolic including reduction of oxidative stress, suppression of oxidized lipids, prevention of ischemic disease, improvement of metabolic syndrome, improvement of impaired glucose tolerance, and abnormal lipid metabolism, comprising the composition according to any one of [1] to [4] above Food for protection and improvement of disorders associated with syndrome, suppression of carcinogenesis, suppression of systemic immune function decline, suppression of vascular endothelial disorders, suppression of arteriosclerosis, prevention of dementia, improvement of brain function, protection of corneal epithelial cells, improvement of dry eye, or suppression of inflammation Composition.
[6] A food comprising the composition according to any one of [1] to [4] above.

[7] Formula I:

[In the formula,
R is hydrogen, ergothionyl, glutathionyl, or cysteinyl]
Oxidative stress reduction, oxidized lipid suppression, ischemic disease prevention, metabolic syndrome improvement, glucose intolerance improvement, improvement of protection and improvement of disorders associated with metabolic syndrome including lipid metabolism abnormality, containing an organic selenium compound represented by as an active ingredient, A pharmaceutical composition for suppressing carcinogenesis, suppressing systemic immune dysfunction, suppressing vascular endothelial damage, suppressing arteriosclerosis, preventing dementia, improving brain function, protecting corneal epithelial cells, improving dry eye, or suppressing inflammation.

[8] The organoselenium compound is of formula II:

The pharmaceutical composition according to claim 7, which is 3-(2-hydroseleno-1H-imidazol-5-yl)-2-(trimethylammonio)propanoate represented by.

[9] Formula III:

Oxidative stress reduction, oxidized lipid suppression, ischemic disease prevention, metabolic syndrome improvement, glucose intolerance improvement, improvement of protection and improvement of disorders associated with metabolic syndrome including lipid metabolism abnormality, containing an organic selenium compound represented by as an active ingredient, A pharmaceutical composition for suppressing carcinogenesis, suppressing systemic immune dysfunction, suppressing vascular endothelial damage, suppressing arteriosclerosis, preventing dementia, improving brain function, protecting corneal epithelial cells, improving dry eye, or suppressing inflammation.

[10] Formula IV:

Oxidative stress reduction, oxidized lipid suppression, ischemic disease prevention, metabolic syndrome improvement, glucose intolerance improvement, improvement of protection and improvement of disorders associated with metabolic syndrome including lipid metabolism abnormality, containing an organic selenium compound represented by as an active ingredient, A pharmaceutical composition for suppressing carcinogenesis, suppressing systemic immune dysfunction, suppressing vascular endothelial damage, suppressing arteriosclerosis, preventing dementia, improving brain function, protecting corneal epithelial cells, improving dry eye, or suppressing inflammation.

The organic selenium compound of the present invention is conventionally known for its antioxidant ability to suppress the generation of peroxides and radicals as an antioxidant based on radical scavenging activity. No evidence of sex was made public. Anti-cancer, anti-arteriosclerosis, etc. are being demonstrated by efficacy tests by administration to human cells and living bodies, and it can be used for new applications such as prevention and improvement of lifestyle-related diseases and diseases caused by aging.

1 shows a chromatogram (A) of selenium 82, a standard selenoneine, and a chromatogram (B) of a selenoneine-containing fish extract obtained in Example 1. FIG. After applying ischemic stress to gerbils, the amount of spontaneous locomotion was measured, the effects of cerebral ischemia-reperfusion were examined, and the effect of the test substance selenium-containing composition (selenoneine-containing fish extract) was evaluated in this model. . After subjecting gerbils to cerebral ischemia-reperfusion, a Y-maze test was performed to examine the effects of cerebral ischemia-reperfusion (day 7). It is the figure which evaluated the effect of. After subjecting gerbils to cerebral ischemia-reperfusion, a Y-maze test was performed to examine the effects of cerebral ischemia-reperfusion (day 14). It is the figure which evaluated the effect of. FIG. 2 shows a study of the effect of ingestion of a selenium-containing composition on improving metabolic syndrome in mice with glucose metabolism disorders. FIG. 2 shows a study of the effect of ingestion of a selenium-containing composition on improving damage associated with metabolic syndrome in mice with glucose metabolism disorders. FIG. 2 is a diagram showing the effect of ingestion of a selenium-containing composition on anticancer effects and inhibition of systemic immune function decline using a colorectal cancer primary tumor model. FIG. 10 is a diagram showing the effect of suppressing systemic immunocompetence decline by ingestion of a selenium-containing composition using a colorectal cancer primary tumor model. FIG. 2 shows a study of the effect of ingestion of a selenium-containing composition on preventing cancer recurrence based on suppression of Treg cells. FIG. 2 is a diagram showing the effect of suppressing the promotion of atherosclerosis due to ingestion of a selenium-containing composition based on suppression of migration of smooth muscle cells. FIG. 10 is a diagram examining the effect of ingestion of a selenium-containing composition that protects cells from amyloid β toxicity. FIG. 4 is a diagram examining changes in the number of viable cells after drying stress due to ingestion of a selenium-containing composition. FIG. 2 shows a study of changes in mucin gene expression due to ingestion of a selenium-containing composition. FIG. 4 shows a study of the effect of ingesting a selenium-containing composition on suppressing NFκ-B activation.

TECHNICAL FIELD The present invention relates to a selenium-containing composition containing an organic selenium compound as an active ingredient for improving metabolic syndrome.

(1) Organic Selenium Compound The selenium-containing composition of the present invention can be obtained, for example, according to the method described in Japanese Patent No. 5,669,056. Unless otherwise indicated herein, an "organoselenium compound" refers to the selenoneine monomer represented by Formula II and its tautomer represented by Formula III, the selenoneine dioxydioxide represented by Formula IV. It is intended that the mers and chemical modifications of selenoneine represented by Formula I are shown and may include "selenium-containing compositions" containing these compounds. Here, the organic selenium compound can be obtained by extracting a biological sample such as fish with an aqueous solvent or an organic solvent to obtain a selenium concentrate, followed by separation and purification by chromatography. Here, "selenium concentrate" refers to a selenium concentrate containing an organic selenium compound (described later) represented by the chemical formulas I to IV of the present invention, and after extracting a sample with an organic solvent or water, and the like obtained by concentrating with. When the selenium concentrate is in the form of a solution, it preferably contains 5 μg/mL or more of the organic selenium compound of the present invention, and is more preferably a dry powder obtained by concentration under reduced pressure.

The organoselenium compounds of interest in the present invention are limited to compounds of Formulas I-IV. Formula I:

and the substituent R is hydrogen, ergothioneine, glutathione, cysteine, acetylcysteine, homocysteine, methylmercury, and an "organic selenium compound" bound to a thiol compound presumed to be produced in vivo. , and also includes metals and polymer materials in which the "organoselenium compound" is bonded via a selenol group.

In one embodiment, the organoselenium compounds of the present invention have formula II:

3-(2-hydroseleno-1H-imidazol-5-yl)-2-(trimethylammonio)propanoate of the formula III:

3-(2-selenoxo-2,3-dihydro-1H-imidazol-4-yl)-2-(trimethylammonio)propanoate represented by As described above, both the organic selenium compounds represented by Formula II and Formula III are monomers of organic selenium compounds. The organic selenium compound of Formula II has a molecular structure in which a selenol group is attached to the carbon atom at the 2-position of the imidazole ring, and the selenol group forms a tautomer in equilibrium with the selenoketone group. , in a solution state, it has two chemical forms, a selenol type (formula II) and a selenoketone type (formula III), depending on the solvent conditions. In the presence of a non-polar solvent where the equilibrium is tilted towards the selenol isomer, the oxidized dimer is readily formed. have a tendency to

Additionally, the organoselenium compounds of the present invention include Formula IV:

of the oxidized dimer represented by (3,3′-(2,2′-diselandiylbis(1H-imidazole-5,2-diyl))bis(2-(trimethylammonio)propanoate)) Also included is a dimer in which a compound having a molecular structure in which a selenol group and a trimethylammonium group are bonded to an imidazole ring is used as a basic unit, and this compound forms a diselenide via a selenol group.

According to the present invention, the selenium content contained in the selenium-containing composition can be determined by fluorescence methods using 2,3-diaminonaphthalene (DAN) (e.g. JH Watson, Anal. Chem., 38, 92-97 ( 1966)), or by HPLC-ICP-MS analysis using a GPC column (see, for example, H. Ge, et al., Anal. Commun., 33, 279-281 (1996)).

In another embodiment, the selenium-containing composition of the present invention may be provided in the form of a composition containing, for example, a compound represented by Formula II (“selenoneine”). The composition containing selenoneine of the present invention may be in the form of, but not limited to, a tablet, paste or beverage containing 0.1 to 100 μg of selenoneine concentrate, dried selenoneine or purified selenoneine as selenium. . When used as food such as supplements and beverages, it is desirable to mold the selenium into a shape suitable for taking the required amount of selenium per day. Other food materials and food additives such as sweeteners, flavors, excipients, thickeners and preservatives may be included.

Raw materials derived from selenoneine, which are typical examples of the organic selenium compound of the present invention, include, for example, tuna (bluefin tuna, southern bluefin tuna, yellowfin tuna, bigeye tuna, albacore), marlin (swordfish, marlin, sailfish, black marlin), bonito ( Skipjack tuna, Marsou tatsuo, Hirasou tatsuo, Hagatsuo, Suma), Mackerel (Chubby mackerel, Scalloped mackerel, Norwegian mackerel, Nijo mackerel, Japanese mackerel), Yellowtail, Horse mackerel (Japanese horse mackerel), Sardines (Sardine, Japanese anchovy), Sea bream (Red sea bream) including fish, marine mammals including toothed whales, yeast (Schizosaccharomyces pombe) and other microorganisms ("Methylmercury and selenium in marine products" Chemistry and Biology 50 (11), 807-817, 2012-11-01; Ｇｅｎｅｔｉｃ ａｎｄ Ｍｅｔａｂｏｌｏｍｉｃ Ｄｉｓｓｅｃｔｉｏｎ ｏｆ ｔｈｅ Ｅｒｇｏｔｈｉｏｎｅｉｎｅ ａｎｄ Ｓｅｌｅｎｏｎｅｉｎｅ Ｂｉｏｓｙｎｔｈｅｔｉｃ Ｐａｔｈｗａｙ ｉｎ ｔｈｅ Ｆｉｓｓｉｏｎ Ｙｅａｓｔ， Ｓ． ｐｏｍｂｅ， ａｎｄ Ｃｏｎｓｔｒｕｃｔｉｏｎ ｏｆ ａｎ Ｏｖｅｒｐｒｏｄｕｃｔｉｏｎ Ｓｙｓｔｅｍ， ＰＬｏＳ Ｏｎｅ．， ２０１４ Ｊｕｌｙ ３１； ９（７）： ｅ１０５１７７参照）。

(2) Selenium-Containing Composition The present invention provides a selenium-containing composition containing the above organic selenium compound as an active ingredient. The content of the organic selenium compound contained in the composition of the present invention may range from 0.000001 to 99% by weight relative to the total weight of the composition. The content of the selenium compound can be adjusted as appropriate. Moreover, when a selenium-containing composition is used as an active ingredient, as described above, it can take a form containing 0.1 to 100 μg of selenium. In addition, the raw material of the composition is not particularly limited as long as it contains selenoneine. Products can also be raw materials.

The organic selenium compound of the present invention may also contain other ingredients in addition to the organic selenium compound as an active ingredient, depending on the purpose. Other ingredients that can be included in the selenium-containing composition of the present invention include, but are not limited to, water; alcohol; processed meat; Food materials and their powders; sugars such as starch, starch syrup, lactose, glucose, fructose, sucrose, mannitol; proteins such as fish powder, egg whites/dried whole eggs, fish eggs, fish proteins, vegetable proteins, collagen, myosin, gluten, etc. common food additives such as spices, sweeteners, edible oils, vitamins; surfactants; excipients; coloring agents; preservatives; coating aids; oils; humectants; thickeners; preservatives; fragrances; and combinations thereof. The compositions of the invention may also contain other agents as desired. The content of such other ingredients and/or other drugs is not particularly limited and can be appropriately selected by those skilled in the art.

(3) Foods, Food Compositions and Pharmaceutical Compositions Containing Selenium-Containing Compositions According to the present invention, foods, food compositions and pharmaceutical compositions containing the composition of the present invention are provided. When the composition of the present invention is used as a food or food composition, it may contain a food-acceptable carrier and other additives according to the desired product form. Such carriers and additives include, for example, excipients, binders, flavoring agents, buffering agents, thickening agents, coloring agents, stabilizers, emulsifying agents, dispersing agents, suspending agents, disintegrating agents, and lubricants. , preservatives and the like. The pharmaceutical composition of the present invention can be used in oral forms such as solid formulations such as foods, food additives, tablets, powders, fine granules, granules, capsules, pills, sustained release formulations, solutions, Forms of liquid preparations such as suspensions and emulsions are included. These can be formulated or commercialized together with carriers and additives, if necessary, by methods commonly used in the art.

(4) Applications The selenium-containing composition containing the selenium compound formulas I to IV of the present invention as an active ingredient, and the food and food composition containing the composition, are useful for reducing oxidative stress, suppressing oxidized lipids, preventing ischemic disease, preventing metabolic Improvement of syndrome, improvement of glucose intolerance, improvement of protection for disorders associated with metabolic syndrome including abnormal lipid metabolism, suppression of carcinogenesis, suppression of decline in systemic immune function, suppression of vascular endothelial disorder, suppression of arteriosclerosis, prevention of dementia, improvement of brain function, corneal epithelium Cell protection, dry eye improvement effect and inflammation suppression are expected. The mode of use as food is not limited, but it may be in the form of health food, functional food, food for specified health use, food with nutrient function claims, nutritional supplement, food with function claims, beverage, and the like. As used herein, "amelioration" means amelioration or alleviation of a disease, symptom or condition, prevention or delay of worsening of a disease, symptom or condition, or reversal, prevention or delay of progression of a disease, symptom or condition. Say. "Reduction" refers to delaying the appearance of, reducing the frequency of, or reducing the severity of, a disease, symptom or condition, and may be used synonymously with "amelioration." In addition, "inhibit" means to completely or partially suppress a disease, symptom or condition. As used herein, the terms "improvement,""reduction," and "inhibition" may be used interchangeably. "Prevention", on the other hand, refers to preventing or delaying the onset of a disease or condition in an individual, or reducing an individual's risk of developing a disease or condition. "Protection" refers to preventing the development of various clinical symptoms, and may be understood to include "prevention".

In order to obtain the above-described desired effects in the selenium-containing composition of the present invention, the amount of the organic selenium compound, which is an active ingredient, is 0.1 ng to 10 mg, more preferably 1 ng to 5 mg, more preferably 1 ng to 5 mg per 1 kg body weight of an adult. is preferably 2 ng to 3 mg, even more preferably 3 ng to 1 mg, still more preferably 5 ng to 500 μg, even more preferably 5 ng to 100 μg, most preferably 10 ng to 50 μg. More specifically, administration or intake of 2 ng to 2550 μg per day is preferable, more preferably 5 ng to 47 μg, still more preferably 10 ng to 14.5 μg. In addition, in the present invention, this amount of active ingredient is divided into 1 to several times a day, and the compound is in the form as it is or in a desired form such as food, composition, medicine, food and drink, It may be administered or ingested.

a. Preventive Effect on Ischemic Diseases and Reduction of Oxidative Stress It is known that in ischemia, a large amount of active oxygen is produced during blood reperfusion, resulting in injury. By ingestion of the selenium-containing composition of the present invention, the bioantioxidant effect is enhanced, oxidative stress in the brain is reduced, and ischemic brain disease can be prevented. For such ischemic disease preventive effect and oxidative stress reducing effect, for example, after administering a selenium-containing composition, a cerebral ischemia-reperfusion model test using gerbils is conducted to confirm the effect of reducing ischemia-reperfusion injury. be able to. More specifically, gerbils are administered a selenium-containing composition (eg, selenoneine-containing fish extract) for 2 weeks, and blood flow in both common carotid arteries is stopped for 5 minutes before reperfusion. Thereafter, administration of the organic selenium compound is continued for 2 weeks, and motor activity and working memory ability can be tested and evaluated (see Example 2 below). In addition, the brain and heart may be removed after sacrifice and the ischemic area biochemically confirmed. INDUSTRIAL APPLICABILITY The selenium-containing composition of the present invention can be used, for example, as a food raw material and as an active ingredient of a composition that alleviates oxidative stress caused by oxygen deficiency and protects against disorders caused by ischemic heart disease, cerebral infarction, and the like.

b. Effect of protection and improvement from metabolic syndrome and disorders associated with it In recent years, the number of patients with non-alcoholic hepatitis has been increasing in addition to hepatitis caused by excessive alcohol intake, viruses, and the like. Ingestion of the selenium-containing composition of the present invention can suppress lipid peroxide (oxidized LDL) and prevent non-alcoholic hepatitis, fatty liver, impaired glucose tolerance, metabolic syndrome and diabetes associated therewith. As an animal model, non-alcoholic hepatitis model mice with diabetes mellitus were prepared by administering streptozotocin and a high-fat diet, and the selenium-containing composition of the present invention was mixed with the diet and administered to induce non-alcoholic hepatitis. It is possible to confirm the preventive effect against oxidative stress in the liver and liver damage associated with For example, the fish extract containing selenoneine, which is the selenium-containing composition of the present invention, and a high-fat diet are ingested, the liver is removed after 4 weeks, and the hepatitis score and the like are measured to determine the oxidative stress caused by the selenium-containing composition of the present invention. reduction of fat accumulation in the liver, development of hepatitis and metabolic syndrome, and protective improvement effects from disorders associated therewith can be evaluated (see Example 3 below).

c. Effect of Preventing Carcinogenesis and Suppressing Effect of Decreased Systemic Immunity By ingesting the selenium-containing composition of the present invention, the effect of suppressing cancer proliferation, metastasis and carcinogenesis and the effect of suppressing decrease of systemic immunity can be enhanced. By using an animal model, it is possible to confirm the effect of preventing carcinogenesis and the effect of suppressing systemic immunocompetence. The growth inhibitory effect on cancer cells is measured by measuring the reduction in tumor area when cancer cells are transplanted into mice and, for example, a feed containing the selenium-containing composition of the present invention (e.g., selenium concentration of 3 mg/kg) is administered. can be confirmed by

The effect of suppressing mutagenicity by the selenium-containing composition of the present invention can be confirmed by a reverse mutation test using bacteria. In addition, under conditions that are closer to living organisms, mammalian-derived cells (eg, Bhas42 cells) can be used to confirm the effect of suppressing transformation by the selenium-containing composition of the present invention. This method is a test method used to detect carcinogens established by the Food and Drug Research Center, and it is possible to distinguish between initiation activity and promotion activity of carcinogenesis. One day after seeding the cells, the transformation inducer 3-methylcholanthrene is added to the medium and treated for 3 days. The medium is replaced with a selenoneine-containing fish extract-added medium, cultured up to 21 days after cell seeding, and then transformed foci are counted. Since the effect can be confirmed for each stage of cancer initiation, promotion, and progression, it is possible to evaluate whether it can be used as a food ingredient that has a cancer-preventing effect (see Example 4 described later). ).

In addition, the selenium-containing composition of the present invention may be administered to mice with bile cancer to confirm the effect of improving systemic immune function. Regarding suppression of systemic immunocompetence, it is possible to verify the carcinogenesis-inhibiting effect of selenium-containing compositions by analyzing regulatory T (Treg) cells that have an immunosuppressive function and promote tumor growth (described later. See Example 4). Ingestion of the selenium-containing compositions of the present invention can improve immune competence in diseased subjects, inhibit cancer growth, or prevent cancer recurrence.

d. Effect of Inhibiting Arteriosclerosis Oxidized LDL is considered to be a part of the mechanism of progression of arteriosclerosis, and it has been reported that smooth muscle cells migrate due to oxidized LDL. It was found that the selenium-containing composition of the present invention has an arteriosclerosis-preventing effect that prevents smooth muscle cells from dedifferentiating and migrating into the vascular lumen, which cause stenosis when arteriosclerosis develops. By administering the selenium-containing composition of the present invention to smooth muscle cells, the effect of inhibiting migration of smooth muscle cells and suppressing arteriosclerosis can be confirmed. For example, by culturing human aortic smooth muscle cells in a medium supplemented with a selenium-containing composition and measuring the number of migrated cells, it is possible to evaluate whether it can be used as a food raw material having an effect of preventing arteriosclerosis. It is possible (see Example 5 described later). According to the present invention, migration of smooth muscle cells can be suppressed, and, for example, promotion of atherosclerosis can be suppressed. Also, vascular endothelial disorders associated with arteriosclerosis can be improved.

e. Effect of Improving Brain Function Aging, genetic predisposition, and lifestyle-related diseases (diabetes, etc.) are known risk factors for Alzheimer's disease. It was found that the selenium-containing composition of the present invention has a brain function-improving effect that reduces the generation of reactive oxygen species due to aging, diabetes, etc., and prevents the onset of dementia. As used herein, "dementia" means Alzheimer's dementia, Lewy body dementia, cerebrovascular dementia, frontotemporal dementia, and the like. In addition, "brain function" means memory function (for example, working memory, short-term memory), emotion, language function, cognitive function, motor function, learning function, attention function, and the like. In this way, brain functions are diverse, and functional deterioration can be suppressed by protecting ischemia-reperfusion injury and cranial nerves. Moreover, it is known that the risk of depression is increased due to diabetes, and not only dementia but also depression can be prevented by protecting against brain function deterioration due to diabetic disorder. The effect of improving brain function as described above can be obtained, for example, by preparing an Alzheimer's disease model mouse by administering streptozotocin, and confirming the effect of reducing the progress of Alzheimer's disease by administering the selenium-containing composition of the present invention (for example, selenoneine-containing fish extract). can be done. It is known that direct administration of streptozotocin into the brain increases intracerebral oxidative stress, causes accumulation of amyloid β and tau protein, and results in pathology similar to Alzheimer's disease. For model preparation, streptozotocin is administered after ingesting the selenium-containing composition of the present invention for one week. They can then be evaluated by ingesting a selenium-containing composition for two weeks and examining working memory and brain tissue. Furthermore, it is possible to biochemically confirm the amyloid β accumulation-inhibiting action by a test using cells. Ingestion of a selenium-containing composition that has an effect of preventing and improving brain function decline and foods made from it can be expected to have a brain function-protecting effect not only on sick people but also on healthy elderly people (implementation described later). See Example 6).

f. Effect of Improving Dry Eye By ingesting a selenium-containing composition, drying and oxidative stress of corneal epithelial cells are reduced and protected, and a preventive effect of dry eye can be obtained. It is said that there are about 8 to 22 million dry eye patients in Japan. Inflammation and persistent oxidative stress exacerbate dry eye, and it has been reported that mucin levels are reduced and mitochondrial malformation is observed in dry eye model mice (Kawai, M., et al., Sci. Rep. ., vol.3, 2455 (2013)). Human immortalized corneal epithelial cells can be cultured in a medium supplemented with an organic selenium compound (eg, selenoneine-containing fish extract), and can be evaluated by measuring mucin production and the like. Dry eye is not a major disease and is often overlooked, but improvement of dry eye is very important for improving QOL of patients. (see Example 7). By using the selenium-containing composition of the present invention, it is possible to protect corneal cells from desiccation stress and prevent and improve dry eye.

g. Inflammatory Suppressive Effect By ingesting the selenium-containing composition of the present invention, redness, swelling, fever, pain and dysfunction associated with inflammation are suppressed, and rheumatoid arthritis, hemolytic anemia and myasthenia gravis caused by inflammation are prevented. can be done. In a normal person, the immunoregulatory system functions in a well-balanced manner, but when the host's immunoregulatory system becomes abnormal for some reason, autoantibodies that recognize the self's biological components as antigens are produced, resulting in various immune reactions. resulting in fibrosis and functional impairment. Examples of such autoimmune diseases include rheumatoid arthritis, hemolytic anemia, and myasthenia gravis. Immunosuppressants such as azathioprine, cyclophosphamide, pincristin, etc. are used as therapeutic agents for such autoimmune diseases, but it is known that administration of these agents causes a wide variety of side effects. However, most topical anti-inflammatory agents applied directly to the affected area are steroids, and their use has been limited due to their side effects. The organic selenium compound of the present invention can be used as food, has no side effects, and is expected to suppress various inflammatory reactions. The target of the anti-inflammatory effect by ingestion of the selenium-containing composition of the present invention is particularly due to NFκ-B activation, such as allergic diseases, anti-cancer, arthritis, autoimmune diseases, inflammatory bowel diseases, It is effective against systemic lupus erythematosus, ankylosing spondylitis, and celiac disease (see Example 8 below).

The following examples illustrate various aspects of the disclosure. It will be apparent to those skilled in the art that numerous modifications, both to materials and methods, may be made without departing from the scope of this disclosure. All reagents and solvents purchased from commercial suppliers should preferably be further purified by distillation, recrystallization, filtration, etc. to remove contaminating peroxides and radicals.

Example 1: Preparation of organic selenium compound An organic selenium compound was obtained by obtaining an extract containing selenoneine from tuna blood bleached water according to the method described in the previously reported Japanese Patent No. 5669056 (Patent Document 1), and Brix55. to obtain a selenoneine-containing fish extract.

Analysis of Selenoneine Content The selenoneine content of the selenoneine-containing fish extract (Brix 55) obtained in Example 1 was calculated as a selenium equivalent (μgSe/g) based on the following measurement values obtained by LC-ICPMS.

(1) Device configuration Separation column: Ultrahydrogel 120 (Waters)
LC pump: Pu712 (GL Science Co., Ltd.)
Sample injector: 9725i (Rheodyne)
ICPMS: ELAN DRC II (Perkin-Elmer)

(2) LC measurement conditions Mobile phase: 0.1% IGEPAL-CA63, 0.1 M ammonium acetate (pH 5.3)
Flow rate: 1.0 mL/min
Sample injection volume: 0.01 mL

(3) ICP-MS measurement conditions Measurement element: Se
Mass number: 82
RF output (W): 1200
Nebulizer gas (Ar, L/min): 1.0
Auxiliary gas (Ar, L/min): 1.3
Plasma gas (Ar, L/min): 17
Makeup gas (Ar, L/min): 0
Reactive gas (Ar, L/min): 0
Pulse stage voltage (eV): 1050
RPq: 2.5
Stay time (sec/amu): 0.4

(4) Preparation of sample 20 µL of the selenoneine-containing fish extract obtained in Example 1 was mixed with 19 times the volume (380 µL) of ultrapure water (Milli-Q Element, Millipore) and mixed at 12000 rpm for 10 minutes. The centrifuged supernatant was used for HPLC-ICPMS analysis.

(5) Preparation of standard selenoneine A standard selenoneine was purified from swordfish blood muscle by the following method:
(5-1) Frozen swordfish fillets were thawed overnight at 5° C., and blood cells were cut out and collected.

(5-2) 0.1 g of cysteine hydrochloride (Wako Pure Chemical Industries, Ltd.) was added to 100 g of blood muscle, and polytron homogenizer (PT MR 6000, KINEMATICA) 6800 rpm in 10 times the volume of acetonitrile (Wako Pure Chemical Industries, Ltd.). for 15 seconds while cooling with ice water.

(5-3) The pulverized product was centrifuged at 6000×g for 10 minutes, and the supernatant was concentrated under reduced pressure using a rotary evaporator to obtain Concentrate 1.

(5-4) Acetonitrile:tetrahydrofuran (Wako Pure Chemical Industries, Ltd.) (1:1, v/v) mixed solution was added to concentrate 1 to separate the concentrate into two layers.

(5-5) Of the separated two layers, the acetonitrile layer was separated and concentrated with a rotary evaporator under reduced pressure to obtain a concentrate 2.

(5-6) Add 5 ml of cooling water to the concentrate 2, apply to Atlantis (registered trademark) dC18300 angstrom column (19 × 150 mm, Nippon Waters Co., Ltd.) equilibrated with 0.1% acetic acid, 0-50% linear acetonitrile Fraction 1 was collected from 40 ml to 50 ml of elution volume under the gradient.

(5-7) Fraction 1 was concentrated with a rotary evaporator under reduced pressure to obtain Concentrate 3.

(5-8) Concentrate 3 was applied to an Ultrahydrogel (registered trademark) 120 column (7.8 x 300 mm Nippon Waters Co., Ltd.) equilibrated with 0.1% acetic acid and 30% acetonitrile, and the discharge amount was 7.5 ml to 9.0 ml. Fraction 2 was obtained by collecting fractions containing selenoneine up to .

(5-9) Fraction 2 was passed through an Ultrahydrogel (registered trademark) 120 column (7.8 x 300 mm Nippon Waters Co., Ltd.) equilibrated with 0.1% acetic acid, and fractions containing selenoneine were collected and evaporated under reduced pressure using a rotary evaporator. to obtain a selenoneine standard.

(5-10) The total selenium content in the selenoneine standard was measured by the fluorescence method described below and was 0.476 μg/mL.

The selenoneine standard products obtained in (5-1) to (5-9) above contain the compounds represented by formulas (1) to (4), and the compound represented by formula (2). is known to be the main component (University of Tokyo doctoral (agricultural) paper pp17-19 (Yumiko Yamashita, 2012)).

(6) Identification and quantification of selenoneine For the sample of (4) and the selenoneine standard, selenium 82 was measured with the LC-ICPMS device described in (1) to (3) to obtain a chromatogram, and selenoneine was used to obtain a chromatogram. was determined.

(6-1) Selenoneine standard has a peak detected at an elution time of around 10.3 minutes. In addition, signals of unidentified selenium compounds are detected. For the selenoneine standard, the integrated value of all selenium signals was measured from 6 to 11 minutes, and the peak area corresponding to the total selenium injection amount was quantified by the fluorescence method described later. considered.

(6-2) The peak area near the elution time of 10.3 minutes in the obtained chromatogram was identified as selenoneine and read using software (TotalChrom, Perkin-Elmer).

(6-3) Formula (5) below:
X=(A/B)×(1000/C)×D/1000 Formula (5)
(here,
X: selenoneine content in the sample (μgSe/g)
A: Peak area at 10.3 min of sample.
B: Detection area per weight of injected selenium of standard selenoneine (/ngSe)
C: microL volume of injected sample in LC-ICPMS (C=5 this time)
D: Dilution ratio with ultrapure water when the sample was prepared (D=20 this time).

(6-4) Inject 10 microL of selenoneine standard into the LC-ICPMS device, measure the integral value of all selenium signals from 6 to 11 minutes, and calculate the amount of selenium injected (4.76 ng in this case). The value obtained by dividing by the detected area per selenium weight (coefficient C in formula (5))

(6-5) Measurement Results FIG. 1 shows the chromatogram (A) of selenium 82 of the selenoneine standard product and the chromatogram (B) of the selenoneine-containing fish extract obtained in Example 1.

(6-6) In chromatogram (B), a peak (arrow) corresponding to selenoneine dimer (Formula IV) was observed as a major selenium compound at a retention time of 10.6 minutes.
Therefore, it was found that most of the selenium compounds contained in the selenoneine-containing fish extract obtained in Example 1 were mainly composed of the compound represented by Formula IV. For the selenoneine-containing fish extract obtained in Example 1, the calculated selenoneine content was 32.5 mgSe/L.

(7) Total selenium content The selenoneine-containing fish extract obtained in Example 1 and the selenoneine standard products obtained in (5-1) to (5-9) are described in the literature (Fluorometric determination of selenium in nanogram amounts in biological materials). R. Hasunuma, T. Ogawa, Y. Kawanishi, Analytical biochemistry (1982), 126: 242-245) to make all the selenium compounds inorganic. ,3-diaminonaphthalene (DAN) and measuring the fluorescence of 4,5-benzopiaselenol (Se-DAN) resulting from the complex formation reaction with Se(IV). It was calculated as the selenium weight per sample weight (mg/kg) based on the obtained measured value. Specifically, it was measured under the following conditions.

(7-1) 10 mg of the selenoneine-containing fish extract obtained in Example 1 was weighed and wet-ashed in a test tube with 1.5 mL of mixed acid (nitric acid: perchloric acid = 2: 1) at 210 ° C. for 2 hours. 0.25 mL of a saturated ammonium oxalate aqueous solution was added to the resulting solution, and the mixture was heated in a 100° C. water bath for 5 minutes.

(7-2) After cooling the sample obtained in (7-1) with water, 0.25 mL of 6M hydrochloric acid was added and heated in a water bath at 100°C for 30 minutes. 0.25 mL was added, and the pH was adjusted to 1.0 to 1.5 using a 6M sodium hydroxide aqueous solution.

(7-3) 1 mL of 1 mg/mL DAN dissolved in 0.1 M hydrochloric acid was added to the sample obtained in (7-2) and heated at 50° C. for 20 minutes. After cooling with water, it was shaken with 1 mL of cyclohexane and the fluorescence of the cyclohexane layer was measured with an excitation light of 379 nm and a fluorescence wavelength of 521 nm.

(7-4) Prepare a calibration curve using test tubes containing 5, 10, 50, and 100 μL of a 1 mg/L selenium standard solution and an operation blank in which no sample is added in parallel with the sample, and measure the total selenium content in the sample. amount was calculated.

The total selenium content of the selenoneine-containing fish extract obtained in Example 1 was 95.3 mg/kg.

Example 2: Examination of Effect of Reducing Ischemia-Reperfusion Injury After subjecting gerbils to cerebral ischemia-reperfusion, locomotor activity was measured and a Y-maze test was conducted to examine the effects of cerebral ischemia-reperfusion. The effect of the test substance (selenoneine-containing fish extract) was evaluated.

1. Test System 7-week-old gerbils (MON/Jms/Gbs Slc, male) were purchased from Japan SLC, Inc., and after acclimatization for 5 days, their body weights were measured and divided into groups. During the test period, normal feed (CRF-1, Oriental Yeast Co., Ltd.) and water were given ad libitum. The test groups were divided into the following six groups.

The administration start date of the test substance was Day 0, and on Day 14, under isoflurane anesthesia, the common carotid artery was sutured with a thread on the day before ischemia. On the next day, the animal was laid on a hot plate set at 40°C ± 3°C to maintain the body temperature at 37°C, the thread at the incision was cut, the bilateral common carotid arteries were pulled, and the arteries were stopped with a clamp for 5 minutes. After that, the clamp was removed, reperfusion was performed, and the incision was sutured. In the normal group, the bilateral common carotid arteries were exposed under isoflurane anesthesia and the skin incisions were sutured without treatment. To the test substance administration group, 6.2 mL/kg of the test substance was forcibly administered daily during the test period using a polypropylene disposable syringe and a stomach tube. 6.2 mL/Kg of water was similarly administered to the normal group and the control group. Gavages were given once daily for 22 or 29 days. When ischemia-reperfusion is performed, hippocampal cell death causes behavioral abnormalities (spontaneous hyperactivity), learning and memory impairment, and the like. Locomotor activity measurement and Y-maze test were performed to evaluate the protective effect of the test substance against brain injury caused by ischemia-reperfusion.

(1) Measurement of locomotor activity Locomotor activity was measured for 1 hour each on Days 6, 13, 15, 17, 19, 21 and 27 (Day 27 is the second course only), with the test substance administration start date being Day 0. rice field. For the measurement, a multi-channel locomotor activity measurement system Supermex (Muromachi Kikai Co., Ltd.) was used.
(2) Y-maze test In the Y-maze test, animals were placed on Day 21 (1st course) and Day 28 (2nd course) and allowed to move freely for 6 minutes, and the rate of choosing a new arm was measured. was evaluated for memory learning behavior.
(3) Selenoneine measurement At the end of the first course, whole blood was collected from the surviving test subjects of groups 1, 2, and 3 from the abdominal aorta with a heparin-filled syringe under 2.5% isoflurane inhalation anesthesia. He was euthanized by exsanguination. The collected blood was centrifuged (3000 rpm, 10 minutes, 4° C.) to collect plasma and clot separately. The selenoneine content in the clot was analyzed according to the method of Example 1.

2. Results One day after ischemia-reperfusion treatment, locomotor activity increased in control group 2 (“control 22-day group” in FIG. 2) and group 5 (“control 29-day group” in FIG. 2). However, it was significantly suppressed in Group 3 (“Selenoneine-containing fish extract administration 22-day group” in Figure 2) and Group 6 (“Selenoneine-containing fish extract administration 29-day group” in Figure 2), which are test substance administration groups. (Fig. 2). On the other hand, Y-maze tests were performed 7 days and 14 days after the treatment, and the control group showed a decrease in the correct answer rate, but the decrease was significantly suppressed in the selenoneine-containing fish extract administration group. no treatment), and it was confirmed that the test substance has a protective effect against brain damage caused by ischemia-reperfusion (Figs. 3 and 4).

The selenium protein content in the clot was not significantly different between Groups 1, 2 and 3, but the selenoneine content was below the detection limit (0.01 μg/g) in Groups 1 and 2. In contrast, 0.95±0.24 μg/g of selenium was detected in Group 3, and the selenoneine content was significantly increased (p<0.05). This result indicated that administration of the test substance for 22 days accumulated selenoneine in blood cells of gerbils.

Example 3: Investigation of Effect of Improving Protection from Metabolic Syndrome and Disorders Associated with Metabolic Syndrome (1) Effect of Improving Metabolic Syndrome Streptozotocin-administered mice suffering from glucose metabolism disorders similar to type I diabetes were given a high-fat diet. rice field. After a predetermined period of time, the effect of suppressing non-alcoholic hepatitis by administering the selenoneine-containing fish extract was verified using the NAFLD activity score, which is a method capable of comprehensively evaluating liver fat accumulation and the like.

More specifically, 10 female SPF mice (C57BL/6J, Japan SLC) were purchased on the 14th day of pregnancy and habituated. All animals were allowed to give birth spontaneously, and the male mice that were born were used for the preparation of model animals. In addition, weaning was performed at the age of 4 weeks after birth.

Observation of offspring: For mice observed to give birth at the time of delivery observation at 8:00, the date of birth was defined as the day before observation, and for mice observed to give birth at the time of delivery observation at 20:00, the date of birth was defined as the day of observation. , calculated from day 0 after birth. Two-day-old male mice were treated with streptozotocin (Sigma-Aldrich Japan Co., Ltd.) in physiological saline (Japanese Pharmacopoeia, Otsuka Pharmaceutical Factory Co., Ltd.) at a concentration of 10 mg/mL, and 20 μL/head (200 μg/head) was applied to the back. A single dose was administered subcutaneously. After that, the mother animals nursed until weaning. They were weaned at the age of 28±2 days after birth, and thereafter fed with a high-fat diet [High Fat Diet 32 (radiation sterilization, CLEA Japan, Inc.)].

From the date of animal arrival until weaning, both mothers and infants were allowed to freely ingest solid diet CE-2 (sterilized by radiation, CLEA Japan, Inc.). After weaning, the streptozotocin-administered animals were given a high-fat diet High Fat Diet 32 (radiation-sterilized) (CLEA Japan, Inc.) ad libitum.

A single dose of the selenoneine-containing fish extract was 15 mg/kg body weight. The administration volume was 10 mL/kg body weight, and gavage administration was performed using an oral probe. A control group was similarly administered at 10 mL/kg body weight. The amount of liquid to be administered to each individual was calculated based on the body weight on the morning of the day (unit: 0.01 mL). The period was once a day from 5 weeks of age for 28 days. After the administration was completed, the rats were exsanguinated by cutting the abdominal aorta, the liver was excised, the left lateral lobe was divided into 6 equal parts, 2 sections were stained with hematoxylin and eosin, and the NAFLD activity score was calculated.

The NAFLD activity score is obtained by scoring the degree of hepatic steatosis, liver parenchymal inflammation, and hepatocyte damage (balloon-like degeneration), and is used for pathological diagnosis of non-alcoholic hepatitis. In the control group, hepatocyte balloon-like degeneration, macrovesicular and microvesicular lipid droplet deposition, and inflammatory cell infiltration including lymphocytes and neutrophils were observed. The NAFLD Activity score was significantly lower in the selenoneine-containing fish extract administration group than in the control group. Selenoneine was suggested to prevent and improve fat accumulation due to metabolic abnormalities caused by metabolic syndrome, and was considered to have preventive and improving effects on metabolic syndrome (Fig. 5, Table 3).

(2) Improving effect from disorders associated with metabolic syndrome The icv-STZ-induced Alzheimer's dementia model is an animal model of Alzheimer's dementia with a diabetes-like background caused by impaired insulin signaling in the brain. , is a model in which the genetic factor is not the primary factor. Therefore, this model is useful for examining therapeutic effects on sporadic Alzheimer's disease. In this example, for the purpose of examining the effect of selenoneine-containing fish extract, which is a test substance, on Alzheimer's dementia, icv-STZ-induced Alzheimer's dementia model mice were induced from 7 days before the start of model induction. Oral gavage was administered for 21 days up to 14 days after initiation. Behavioral analysis was performed on the 13th day after the start of model induction, and the mice were sacrificed and analyzed on the 14th day after the start of model induction. By comparing a solvent-administered group (RO water, control-administered group) and a selenoneine-containing fish extract-administered group, the effect of the selenoneine-containing fish extract on Alzheimer's dementia in an icv-STZ-induced Alzheimer's-type dementia model was examined.

Mice (C57BL/6J, 6-week old male, Japan SLC Co., Ltd.) were treated with streptozotocin (Sigma-Aldrich Japan Co., Ltd.) in physiological saline (Japanese Pharmacopoeia, Otsuka Pharmaceutical Factory Co., Ltd.) at a concentration of 20 mg/mL. and administered into the lateral ventricle. An icv-STZ-induced Alzheimer's dementia model animal is prepared by administering 3 μL to the left ventricle once on the day of model induction, and administering 3 μL to the right ventricle once on the second day after the start of model induction. did. During the test period, solid diet CE-2 (radiation sterilized, CLEA Japan, Inc.) was allowed to ingest freely, and on the day before the start of administration, 20 male mice were randomly stratified by weight so that the average weight was even. The animals were grouped according to the extraction method into an icv-STZ+control group and an icv-STZ+selenoneine-containing fish extract administration group (10 animals/group). The administration volume of the test substance was 6.2 mL/kg body weight, and gavage administration was performed once a day for 21 days using an oral probe. The control group was similarly administered RO water at 6.2 mL/kg body weight. The amount of liquid to be administered to each individual was calculated based on the body weight on the morning of the day (unit: 0.01 mL).

After the end of the administration period, the spontaneous alternation behavior was analyzed using the Y-maze. One day before the end of dosing, mice were placed in the center of the maze and allowed to explore freely for 5 minutes. After 5 minutes, mice were returned to their home cages and the Y-maze was disinfected with 50% ethanol, followed by subsequent behavioral testing of mice. On the day of the end of dosing, the entrances to two of the three arms were walled off and the mouse was placed in the center of the maze. After the mice entered the open arms, the walls placed at the entrances to the two remaining arms were removed and allowed to explore freely for 8 minutes. During this time, all times the mice entered the arms and the order in which they entered the arms were recorded. After 8 minutes, mice were returned to their home cages and the Y-maze was disinfected with 50% ethanol prior to testing the next mouse. Alternation behavior rate (%) was used as an index of SAB. The alternation behavior rate was calculated by the following formula (Fig. 5, Table 4).
Alternate behavior rate = 100 × (number of times entering different arms three times in a row) / (total number of times entering arms - 2)
As a result, the selenoneine-containing fish extract group had a significantly higher alternation behavior rate than the control (P<0.05). It was suggested that selenoneine intake protects against diabetes-induced decline in brain function.

Example 4: Investigation of anticancer effect or suppressive effect on systemic immune function decrease (1) Verification of anticancer effect and suppressive effect on systemic immune function decrease using colorectal cancer primary tumor model We investigated the effects on tumor growth and systemic immunity in patients using cancer model animals. Each group was fed between 1.73 and 2.65 g per day. The selenoneine-containing fish extract intake obtained from the selenoneine-containing fish extract feed varied between 81.48 and 125.82 mg per day. As a result of using a colorectal cancer primary tumor model, primary tumor growth tends to be suppressed, and the significant attenuation of IL-10 production seen in gallbladder cancer animals was not observed in the selenoneine administration group. Possibility of enhancing immunity was shown. In addition, regulatory T (Treg) cells were reduced by selenoneine administration, suggesting the possibility of improving the regulation of anti-tumor immunity by Treg cells. In other words, it was suggested that selenoneine has the effect of improving the systemic immunity of cancer patients and suppressing tumor growth (Figs. 7 and 8).

BALB/c female mouse-derived colorectal cancer cell Colon Tumor 26 (CT-26) strain (ATCC No. CRL-2638) was mixed with 10% fetal bovine serum (171012, Nichirei Bioscience) containing RPMI-1640 medium (R8758, Sigma-Aldrich company). In addition, mouse splenocytes were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 50 μM 2-mercaptoethanol (21417-52, Nacalai Tesque). Using a water jacket incubator (APC-30D, Astec), cells were cultured under conditions of 37° C., 5% CO ₂ and 100% humidity. Mice (BALB/c females, Charles River) were fed a selenoneine-containing diet or a control diet from 5 to 8 weeks of age (see Table 5 below). After one week of acclimatization (6 weeks old), 0.5 million cells of CT26 strain were subcutaneously transplanted per mouse. Mice were euthanized 14 days after transplantation (8 weeks old), tumors were excised, and tumor volumes were measured. Tumor volume (mm ³ ) was calculated according to the formula of width (mm) squared×length (mm)×0.5. In addition, the spleen was removed, and a subset of CD4-positive Foxp3-positive regulatory T cells (Treg) in the splenocytes was analyzed by flow cytometry using a fluorescent staining cell analyzer (AccuriC6, BD bioscience) and FlowJo software (TreeStar). Measured by The IL-10 concentration in the splenocyte culture supernatant was measured by ELISA.

ＡＩＮ－９３Ｍおよびセレノネイン含有飼料の魚エキス以外の原料はオリエンタル酵母より購入した。

Raw materials other than the fish extract for the AIN-93M and selenoneine-containing feeds were purchased from Oriental Yeast.

Regulatory T (Treg) cells have an immunosuppressive function and promote tumor growth, and it was strongly suggested that Treg cells in gallbladder cancer mice could be suppressed by ingesting a diet containing selenoneine. In addition, the ability to produce IL-10, which regulates antitumor activity through regulatory immunity, was significantly attenuated in the bile cancer mice, but not in the selenoneine-containing diet group. It was suggested that tumor growth was also suppressed. This suggests that selenoneine improves systemic immunity and suppresses cancer growth. Suppression of Treg cells was also considered effective in preventing cancer recurrence (Fig. 9).

(2) Verification of carcinogenic inhibitory effect using Bhas 42 cells By conducting a transformation test using Bhas 42 cells, selenonein, which is the main component of purified selenoneine, is examined for the presence or absence of carcinogenic inhibitory effects in vitro. did. In a dose-finding study, treatment of Bhas 42 cells with purified selenoneine (0.31, 0.63, 1.3, 2.5, 5.0, 10 μM) showed no cytotoxic effects (data not shown). Therefore, based on this result and data from other experiments, the concentration in the transformation test was set at 1 μM. Transformation studies investigated whether the number of transformed foci induced by 1 μg/mL 3-methylcholanthrene (MCA) (Sigma-Aldrich) was reduced by 1 μM selenoneine purified product. That is, the transformation rate (number of transformed foci/well) was compared between the MCA+ultrapure water group and the MCA+purified selenoneine group. As a result, the respective transformation rates were 15.8 and 10.2, indicating a significant decrease in the number of transformed foci. These results suggest that selenoneine has an in vitro carcinogenesis inhibitory effect.

Bhas 42 cells (mouse whole fetal BALB/c 3T3 A31-1-1 cells introduced with v-Ha-ras gene, Food and Drug Safety Center) were detached with 0.25% trypsin, A suspension with a cell concentration of 2000 cells/mL was prepared. 2 mL (4000 cells) of this cell suspension was dispensed onto a plate (6 wells/group) and cultured for 1 day. One day after seeding, the MCA preparation was added and treated for 3 days. Treatment with purified selenoneine and RA (retinoic acid, Sigma-Aldrich) was performed by changing the medium (2 mL per well) 4, 7 and 11 days after seeding. 14 days after seeding, the medium was replaced with fresh medium (10-day treatment) and cultured for an additional 7 days. Plates for transformation test were fixed with methanol 21 days after seeding, stained with 5 vol% Giemsa solution, and transformed foci per well were counted.

The transformation rate of the MCA + selenoneine purified product group (15.8 cells/well) decreased to about 2/3 compared to the transformation rate of the MCA + ultrapure water group (10.2 cells/well), which was significant. showed a decline. This result suggested that selenoneine, which is the main component of purified selenoneine, has an anticancer effect.

Example 5: Examination of Arteriosclerosis Inhibitory Effect Human aortic smooth muscle cells were used to examine the effect of test substances on cell migration. As a result, 2 mg/ml selenoneine-containing fish extract and 100 nM purified selenonein inhibited migration in the presence of collagen.

AoSMC cells (aortic smooth muscle cells) (LONZA, CC-2571) were adjusted to the required number of cells in a special medium (SmGM-2 Bullet Kit (LONZA, CC-3182)). Using a cell migration assay kit (CytoSelect ^™ 96-well Cell Migration Assay (8 μm), Fluorometric, Cosmo Bio, CBA-106), AoSMC cells were subjected to cell migration assay at 1×10 ⁵ cells/0.1 mL/well. The cells were seeded in the upper chamber of the transwell attached to the kit, and a test medium containing the test substance (2 mg/ml selenoneine-containing fish extract or 100 nM purified selenonein) and 300 μg/ml collagen peptide was added to the lower chamber, followed by culturing for 48 hours. After that, the cells were detached and lysed from the transwell membrane with CyQuant GR Dye attached to the cell migration assay kit, and migrated cells were measured.

Addition of purified selenoneine and selenoneine-containing fish extract significantly inhibited cell migration. It was suggested that the promotion of atherosclerosis can be suppressed by suppressing migration of smooth muscle cells (Fig. 10).

Example 6: Investigation of Effect of Improving Brain Function Amyloid β protein, which is considered to be one of the causes of Alzheimer's disease, has neurotoxicity. It is expected to have the effect of preventing dementia by protecting cells from the toxicity of amyloid β. In this example, PC-12 cells were used to verify whether a test substance can protect against amyloid β toxicity.

PC-12 cells (RIKEN BioResource Center, RCB0009, Lot No. 52, 53, 54) were added to 395 ml of 10% FBS, Horse Serum-added DMEM medium (DMEM medium (Nacalai Tesque, Cat. No. 08456-65), penicillin - Prepared by adding 5 ml of streptomycin solution and 50 ml of FBS (Biowest, Cat. No. S1820, Lot No. 516536) and 50 ml of Horse Serum (GIBCO, 16050-122)) in a _CO2 incubator (5% _CO2 , 37°C). Medium changes were performed every other day. A 0.25% Trypsin-EDTA solution was used for cell detachment.

Nerve cells (PC-12) were seeded at 2×10 ³ cells/100 μL/well on a 96-well plate (collagen-coated) in the above DMEM medium, and cultured in a CO ₂ incubator (5% CO ₂ , 37° C.). The next day, the medium was replaced with a medium containing the test substance (containing 1% FBS, 10 ng/mL NGF (Alomone Labs, PRODUCT#N-100)). Amyloid β (1-40) (SIGMA, Cat. No. A1075-.1MG) (PBS (Nissui Pharmaceutical Co., Ltd., Code 05913) to a final concentration of 1 mM after culture for 24 hours. (50 μL/well) was added (50 μL/well) and cultured for an additional 24 hours. After removing the culture supernatant from each well after the completion of the culture, it was replaced with 100 μL (per well) of the test medium containing 10% viable cell counting reagent (WST-8) (Nacalai Tesque, Cat. No. 07553-44). , 30 min and 90 min. Next, the absorbance of each well (absorption wavelength 450 nm, reference wavelength 630 nm) was measured with a microplate reader (Precision microplate reader (Molecular Devices)), and the amount of change in absorbance per hour was calculated from both values. It was determined as a number and the viability of the cells was measured. In this example, selenoneine-containing fish extract: 2 mg/mL and selenoneine: 100 nM were used as test substances (final concentration).

With the addition of amyloid β, the viable cell count was about 70% of that in the non-addition group. When each test substance was treated under these conditions, a significant improvement in cell viability was observed in the selenolein-containing fish extract compared to the amyloid β-added control (Fig. 11).

Example 7: Investigation of dry eye improvement effect HCE-T cells (immortalized human corneal epithelial cells) (Riken BioResource Center, RCB2280) were mixed with test medium (DMEM/F12 (ThermoFisher, Cat. No. 11330-032) (1 : 1), 10% FBS (Biowest, Cat. No. S1820, Lot No. 516536), 1% NEAA (Gibco, Cat. No. 11140-050), 1% antibiotics (Nacalai Tesque, Cat. No. 26253 -84)) was precultured in a T75 flask in a CO ₂ incubator (5% CO ₂ , 37° C.) until the required number of cells was reached. For passaging the cells, a trypsin/EDTA solution (Nacalai Tesque, Cat. No. 32777-44) was used to detach the cells from the flask, neutralize the trypsin with test medium, and then collect the cells by centrifugation. After that, the cells were resuspended again in the test medium and used as a cell suspension.

The selenoneine-containing fish extract is water soluble and was dissolved directly in the medium to make a stock solution and use.

(1) Dry stress (dry eye) avoidance test in corneal epithelial cells HCE-T was seeded at 2.5 × 10 ⁴ cells/0.1 mL/well in a 96-well plate and placed in a CO ₂ incubator (5% CO ₂ ). After culturing at 37°C for 5 hours, it was confirmed that the cells had adhered to the bottom of the plate, and the medium was replaced with a test medium containing the test substance. After that, it was cultured for 1 day. Subsequently, the culture supernatant was removed and the cells were exposed to desiccation (dry stress treatment time: 10 minutes). After that, a dedicated medium was added, and cultured again for 3 hours in a CO ₂ incubator (5% CO ₂ , 37° C.). Proliferation of cells was measured using Viable Cell Count Reagent SF (Nacalai Tesque, Cat. No. 07553-44) (WST-8).

(2) Mucin Gene Expression Analysis Test HCE-T was seeded at 6×10 ⁴ cells/0.5 mL/well in a 48-well plate and cultured in a CO ₂ incubator (5% CO ₂ , 37° C.) for 1 day. , was replaced with test medium containing the test substance. Five hours after the addition of the test substance, total RNA was recovered from the cells, converted to cDNA, and subjected to mucin gene expression analysis by real-time PCR (Light Cycler 96 System (Roche)). Analysis target gene: membrane-bound mucin gene MUC1, internal standard gene: GAPDH.

Addition of the selenoneine-containing fish extract significantly increased the number of viable cells and mucin gene expression after drought stress compared to the control, indicating that the selenium-containing composition of the present invention protects corneal cells from drought stress, It showed the effect of preventing and improving eyes (Figs. 12 and 13).

Example 8: Anti-inflammatory effect A transcription factor called "NFκB" is involved in various inflammatory responses, and when cells are stimulated by cytokines such as TNF-α, inactive NFκB is activated. An inflammatory response is induced. Expression of NFκ-B is reported to be enhanced in many diseases including allergic diseases and cancer (Kagaku to Biology, Vol.47, No.9, P.602-604 (2009) ). The test substance was confirmed to have an anti-inflammatory effect by suppressing the activity of NFκ-B induced by TNF-α. In the NFκ-B activity measurement system, a reporter gene was introduced into target cells, and NFκB transcriptional activity induced by TNFα stimulation was measured by luciferase activity. The final concentrations of the test substances were selenoneine (crude purification): 0.4 mg/mL (HEK293 cells) and selenoneine (purification): 100 nM.

HEK293 cells (RIKEN BioResource Center, RBC1637, Lot.20) were prepared in a basal medium (MEM, 10% FBS, 1% NEAA, containing antibiotics (MEM NEAA: Gibco, Cat. No. 11140-050, FBS: Biowest, Cat.No.S1820, Lot No.516536, Antibiotics: Nacalai Tesque, Cat.No.26253-84), in a T75 flask in a _CO2 incubator (5% _CO2 , 37°C) to the required number of cells. For passaging the cells, a trypsin/EDTA solution (Nacalai Tesque, Cat. No. 32777-44) was used to detach the cells from the flask, neutralize the trypsin with test medium and then centrifuge to Cells were harvested, then resuspended in test medium again and used as a cell suspension.

HEK293 cells were seeded at 1.5×10 ⁴ cells/0.1 mL/well in 96-well plates and cultured in a CO ₂ incubator (5% CO ₂ , 37° C.) for 1 day. Then, a DNA vector (pGL4.32 [luc2P/NF-kB-RE/Hygro] vector (Promega, Cat. No. E8491)) was introduced into cells using a transfection reagent (Roche, Cat. No. 06366244001). The next day, after replacement with test medium (0.1 mL/well) supplemented with a test substance (selenoneine-containing fish extract final concentration: 0.4 mg/mL, or selenoneine final concentration: 100 nM), 37° C., 5% CO ₂ for 2 hours. 5 μL (per well) of 200 ng/mL TNF-α medium (prepared by adding 10 μL/mL TNF-α (SIGMA, Cat. No. T0157-10UG) stock to test media) was added to give a final concentration of TNF-α : added to 10 ng/mL. After that, the cells were cultured at 37° C. and 5% CO ₂ for 5 hours, and TNF-α stimulation activated NF-κB and promoted luciferase gene induction. After culturing for 5 hours, 30 μL of culture supernatant was removed from each well. 70 μL of luciferase assay reagent (Promega, Cat. No. E2610) was added to each well (total 150 μL of culture supernatant), gently mixed, and allowed to stand for 2 minutes. After that, the relative luminescence intensity (RLU: Relative Light Units) of each well was measured using a plate reader for luminescence measurement (Luminometer: Varioscan Flash, Model No. 5250040, Thermo Fisher Scientific). 200 mM ammonium pyrrolidine dithiocarbamate (APDC) (SIGMA, Cat. No. P8765-1G) was used as a positive control.

Both selenoneine and selenoneine-containing fish extract significantly inhibited NFκ-B activation by addition of TNF-α. It suppressed inflammatory reactions involved in various diseases such as allergy, arthritis, rheumatism and cancer, and chronic inflammatory conditions (Fig. 14).

From the above, it will be appreciated that although specific embodiments of the disclosure have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the disclosure. sea bream. Accordingly, the disclosure is not limited except as by the appended claims. Further, the claims are entitled to their full scope and equivalents.

Example 9: Examination of Effect of Improving Impaired Glucose Tolerance Conventionally, the expression level of the selenoprotein P (SELENOP) gene in the human liver correlates with insulin resistance. Impairment and induction of abnormalities in glucose metabolism, and conversely, knockout of the SELENOP gene and knockdown by RNAi have been reported to improve whole body insulin sensitivity and glucose tolerance in mice (Misu H. , et al., Cell Metabolism, 12, 483-495 (2010)). 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) is also a positive regulator of glycolysis and insulin signaling, and inhibition of PFKFB3 with inhibitors results in stimulation with insulin. Inhibition of glucose uptake and translocation of glucose transporter (GLUT4) to the cell membrane, and Akt signaling system, and conversely, activation of Akt signaling system in cells overexpressing the PFKFB3 gene. (Trefely S., et al., J. Biol. Chem., 290, 25834-25846 (2015)).

In this example, the effect of the selenoneine-containing fish extract on the expression levels of the SELENOP gene and the PFKFB3 gene was examined. Normal human fibroblasts (Kurabo, KF-4109) were added to a special medium [DMEM (Nacalai tesque, Cat. No. 08456-65), 10% FBS (Biowest, Cat. No. S1820, Lot No. 516536), 1% Penicillin-Streptomycin Mixed Solution (Nacalai tesque, Cat. No. 26253-84)] in a CO ₂ incubator (5% CO ₂ , 37° C.) to the number of cells used and added to 8×10 ⁵ cells in 8 cm diameter Petri dishes. Cells were cultured after seeding at cells/15 mL. The next day (80% confluent), the medium was replaced with 15 mL medium containing (1 mg/mL) or not selenoneine-containing fish extract, and cultured for 24 hours. After culturing, the cells were detached from the wells using a 2.5 g/l-Trypsin/1 mmol/l-EDTA solution (Nacalai tesque, Cat. No. 32777-44). Specifically, the culture supernatant of each well was removed, 0.3 mL/well of 1×PBS (Nissui Pharmaceutical Co., Ltd., Code 05913) was added, the cells were washed, and after removing the PBS, 50 μL of 2. A 5 g/l-Trypsin/1 mmol/l-EDTA solution was added and reacted at 37°C. After that, 450 μL of PBS containing 10% FBS was added to each well to stop the trypsin reaction. The cell detachment solution was recovered, centrifuged at 500 xg for 10 minutes at room temperature, the supernatant was removed, and the cells (precipitate) were recovered. Total RNA was collected from cells using RNeasy Mico Kit (QIAGEN, Cat. No. 74004), and RNA concentration and purity were measured with a spectrophotometer and bioanalyzer (Agilent Technologies, Agilent 2100). Subsequent operations involved amplification, fluorescent labeling, and quality check of total RNA samples using Affimetrix products such as the GeneChip WT Terminal Labeling and Controls Kit according to their instruction manuals. The conditioned samples were then hybridized to DNA microarrays (Affimetrix, GeneChip Human Genome U133 Plus 2.0 Array) using a GeneChip Scanner 3000 7G System (Affimetrix), washed and stained, and post-hybridization DNA microarray was scanned, the image file was converted into numerical data, and processing such as background subtraction, normalization, and expression ratio calculation was performed.

Since the selenoneine-containing fish extract suppressed the expression level of the SELENOP gene to 1/3 or less and increased the expression level of the PFKFB3 gene to more than twice that of the non-additive (control), the selenium-containing composition of the present invention was shown to have an effect of improving impaired glucose tolerance (Table 7).

Example 10: Investigation of Effect of Improving Abnormal Lipid Metabolism 27-Hydroxycholesterol (27HC) is a metabolite of cholesterol abundantly present in the blood, promotes the development of arteriosclerosis, and induces inflammation in blood vessels. 27HC in the body is catabolized by cytochrome P450 family 7 subfamily B member 1 (CYP7B1) (Umetani M. et al., Cell Metabolism, 20, 172-182 (2014)). In fact, CYP7B1 gene-knockout mice show a 4- to 5-fold increase in blood and tissue 27HC concentrations compared to wild-type mice (Umetani M., supra). In this example, the effect of the selenoneine-containing fish extract on the expression level of the CYP7B1 gene was examined. Cells and samples were prepared and data collected and analyzed in the same manner as in Example 9. The selenoneine-containing fish extract increased the expression level of the CYP7B1 gene by 8 times or more compared to the additive-free (control), indicating that the selenium-containing composition of the present invention has an effect of improving dyslipidemia ( Table 8).

The selenium-containing composition of the present invention can be newly used as an agent for reducing ischemia-reperfusion injury, an anti-metabolic syndrome agent, and the like. A new application of selenoneine concentrate extracted from processed marine product residue has been found, and it can contribute to increasing the added value of underutilized marine products. It is possible to commercialize new ingredients and functional materials that meet the health consciousness of consumers.

All publications and patent documents cited herein are hereby incorporated by reference in their entirety. Although specific embodiments of the invention have been described herein for purposes of illustration, it will be appreciated by those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. will be easily understood.

Claims (12)

Formula I:

[In the formula,
R is hydrogen, ergothionyl, glutathionyl, or cysteinyl]
Containing an organic selenium compound represented by as an active ingredient, suppression of colorectal cancer cell growth, suppression of colorectal cancer formation, prevention of non-alcoholic hepatitis, prevention of Alzheimer's dementia, accompanied by NFκ-B activation A composition for suppressing inflammation, protecting corneal epithelial cells by activating membrane-bound mucin gene MUC1, suppressing expression of selenoprotein P gene, improving impaired glucose tolerance, and inducing expression of CYP7B1 gene. The organic selenium compound has the formula II:

The composition according to claim 1, which is 3-(2-hydroseleno-1H-imidazol-5-yl)-2-(trimethylammonio)propanoate represented by: Formula III:

Containing an organic selenium compound represented by as an active ingredient, suppression of colorectal cancer cell growth, suppression of colorectal cancer formation, prevention of non-alcoholic hepatitis, prevention of Alzheimer's dementia, accompanied by NFκ-B activation A composition for suppressing inflammation, protecting corneal epithelial cells by activating membrane-bound mucin gene MUC1, suppressing expression of selenoprotein P gene, improving impaired glucose tolerance, and inducing expression of CYP7B1 gene. Formula IV:

Containing an organic selenium compound represented by as an active ingredient, suppression of colorectal cancer cell growth, suppression of colorectal cancer formation, prevention of non-alcoholic hepatitis, prevention of Alzheimer's dementia, accompanied by NFκ-B activation A composition for suppressing inflammation, protecting corneal epithelial cells by activating membrane-bound mucin gene MUC1, suppressing expression of selenoprotein P gene, improving impaired glucose tolerance, and inducing expression of CYP7B1 gene. The composition according to any one of claims 1 to 4, wherein the organic selenium compound is contained in an amount of 5 ng to 500 µg/kg body weight of an adult, which is administered or taken once to several times a day. Suppression of colon cancer cell growth, suppression of colon cancer formation, prevention of non-alcoholic hepatitis, prevention of Alzheimer's disease, NFκ-B, comprising the composition according to any one of claims 1 to 5 A food composition for suppressing inflammation associated with activation, protecting corneal epithelial cells by activating membrane-bound mucin gene MUC1, suppressing expression of selenoprotein P gene, improving impaired glucose tolerance, and inducing expression of CYP7B1 gene . Suppression of colon cancer cell growth, suppression of colon cancer formation, prevention of non-alcoholic hepatitis, prevention of Alzheimer's disease, NFκ-B, comprising the composition according to any one of claims 1 to 5 A food for suppressing inflammation associated with activation, protecting corneal epithelial cells by activating membrane-bound mucin gene MUC1, suppressing expression of selenoprotein P gene, improving impaired glucose tolerance, and inducing expression of CYP7B1 gene . Formula I:

[In the formula,
R is hydrogen, ergothionyl, glutathionyl, or cysteinyl]
Containing an organic selenium compound represented by as an active ingredient, suppression of colorectal cancer cell growth, suppression of colorectal cancer formation, prevention of non-alcoholic hepatitis, prevention of Alzheimer's dementia, accompanied by NFκ-B activation A pharmaceutical composition for suppressing inflammation, protecting corneal epithelial cells by activating membrane-bound mucin gene MUC1, suppressing expression of selenoprotein P gene, improving impaired glucose tolerance, and inducing expression of CYP7B1 gene. The organic selenium compound has the formula II:

The pharmaceutical composition according to claim 8, which is 3-(2-hydroseleno-1H-imidazol-5-yl)-2-(trimethylammonio)propanoate represented by. Formula III:

Containing an organic selenium compound represented by as an active ingredient, suppression of colorectal cancer cell growth, suppression of colorectal cancer formation, prevention of non-alcoholic hepatitis, prevention of Alzheimer's dementia, accompanied by NFκ-B activation A pharmaceutical composition for suppressing inflammation, protecting corneal epithelial cells by activating membrane-bound mucin gene MUC1, suppressing expression of selenoprotein P gene, improving impaired glucose tolerance, and inducing expression of CYP7B1 gene. Formula IV:

Containing an organic selenium compound represented by as an active ingredient, suppression of colorectal cancer cell growth, suppression of colorectal cancer formation, prevention of non-alcoholic hepatitis, prevention of Alzheimer's dementia, accompanied by NFκ-B activation A pharmaceutical composition for suppressing inflammation, protecting corneal epithelial cells by activating membrane-bound mucin gene MUC1, suppressing expression of selenoprotein P gene, improving impaired glucose tolerance, and inducing expression of CYP7B1 gene. The pharmaceutical composition according to any one of claims 8 to 11, wherein the organic selenium compound is contained in an amount of 5 ng to 500 µg/kg body weight of an adult, which is administered or taken once to several times a day.

Images