KR20190080877A - Organoselenium compound-containing composition - Google Patents

Abstract

It is an object of the present invention to examine the physiological activity of selenonine, an organic selenium compound, and discover new uses thereof. According to the present invention, there is provided a composition containing an organic selenium compound as an active ingredient, a food containing the composition, a food composition, and a medicinal composition. By ingesting the composition of the present invention and the like, it is possible to improve the protection of disorders accompanied by metabolic syndrome including oxidative stress reduction, lipid oxidation inhibition, prevention of ischemic diseases, improvement of metabolic syndrome, improvement of glucose tolerance abnormality and lipid metabolism abnormality, It is expected to have a remarkable effect on inhibition of depression, inhibition of vascular endothelial dysfunction, inhibition of arteriosclerosis, prevention of dementia, improvement of brain function, protection of corneal epithelial cells, improvement of dry eye, or inhibition of inflammation.

Description

Organoselenium compound-containing composition

The present invention relates to a composition containing an organic selenium compound as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition for preventing or ameliorating oxidative stress and ischemic diseases, improvement of fatty liver, improvement of glucose tolerance, improvement of metabolic syndrome including lipid metabolism abnormality, improvement of protection of disorders accompanying metabolic syndrome, A food composition, a food composition, and a medicinal composition for the prevention of arteriosclerosis, prevention of dementia and improvement of brain function, protection of corneal epithelial cells and improvement of dry eye and / or inhibition of inflammation.

It has been found that selenoneine, an organic selenium compound, has a strong antioxidant activity (radical scavenging activity) and shows a biological antioxidative effect on human cells (Patent Document 1). A substance having an antioxidant activity removes reactive oxygen species in the living body and exhibits various physiological activities. Since selenium is an essential trace element in the human body, intake of selenonine, which is an organic selenium derived from fish species, can compensate for the shortage of selenium ingestion from the meal. Therefore, application to functional foods containing selenonine as an active ingredient is expected.

It is possible to think that selenonine contributes to prevention of this lifestyle-related disease and anti-aging because the prevention effect and the anti-aging effect on lifestyle diseases such as cancer and diabetes are known in fish meal, The interaction between selenonine and other biomolecules has not been verified. In particular, the effect of increasing the antioxidant activity, such as the radical scavenging activity, by the selenonine, which is taken into the human body or animal cells by the ingestion of selenonine, has not been investigated so far. Therefore, it was necessary to verify the physiological activity of selenonine against biomolecules, model cells, and living organisms, and to verify a wide range of validity for the development and practical use of a new use of selenonine.

Patent Document 1: Japanese Patent No. 5669056

The present invention relates to a health functional study of selenonin, an organic selenium compound, which analyzes the effectiveness of a selenone compound in a living body using mammalian cells and rodents to prevent the development of anti-metabolic syndrome, prevention of cancer, prevention of ischemic diseases, prevention of dementia, , Inflammation inhibition and the like, which are caused by the in vivo oxidative stress disorder. Accordingly, there is provided a selenium compound-containing composition having novel food functionalities such as antineoplastic syndrome action containing selenonine.

Oxidative stress is defined as showing an undesirable state in a living body due to a collapse of the balance between the oxidation reaction and the antioxidation reaction of the living body caused by active oxygen, active nitrogen species or free radicals. The generation and oxidation of active oxygen and free radicals Stress is involved in a wide range of diseases such as inflammation, arteriosclerosis, cancer, aging, neural diseases, respiratory diseases, cataracts, skin diseases, digestive diseases, heart diseases and hypertension, It is known that it plays a central role. Selenonine having a strong radical scavenging activity reduces active oxygen, free radicals, and oxidative stress generated in vivo and verifies the preventive effect of the diseases caused by these oxidative stress, Can be developed.

The inventors of the present invention found that the intake of a selenium compound-containing composition has a preventive effect of a stress-relieving and stress-induced disorder that protects against cerebral disorders caused by cerebral ischemia, by biochemically analyzing the health function of selenonin The present invention has been completed.

That is, the present invention is as follows.

[1] Formula I:

Wherein R is hydrogen, an erthioyl group, a glutathionyl group, or a systeinyl group,

The present invention relates to a method for the prevention of metabolic syndrome including prevention of oxidative stress, prevention of oxidative lipid, prevention of ischemic diseases, improvement of metabolic syndrome, improvement of impaired glucose tolerance, prevention of metabolic syndrome including metabolic syndrome, A composition for inhibiting reduction of systemic immunity, inhibition of vascular endothelial dysfunction, inhibition of arteriosclerosis, prevention of dementia, improvement of brain function, protection of corneal epithelial cells, improvement of dry eye, or inhibition of inflammation.

[2] Organic selenium compound: Formula II:

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

[3] Formula III:

As an active ingredient, an effective amount of an organic selenium compound represented by the following formula (1): (1) an oxidative stress reduction, a lipid oxidation inhibition, an ischemic disease prevention, a metabolic syndrome improvement, an impaired glucose tolerance, A composition for inhibiting reduction of systemic immunity, inhibition of vascular endothelial dysfunction, inhibition of arteriosclerosis, prevention of dementia, improvement of brain function, protection of corneal epithelial cells, improvement of dry eye, or inhibition of inflammation.

[4] Formula IV:

The present invention relates to a method for the prevention of metabolic syndrome including prevention of oxidative stress, prevention of oxidative lipid, prevention of ischemic diseases, improvement of metabolic syndrome, improvement of impaired glucose tolerance, prevention of metabolic syndrome including metabolic syndrome, A composition for inhibiting reduction of systemic immunity, inhibition of vascular endothelial dysfunction, inhibition of arteriosclerosis, prevention of dementia, improvement of brain function, protection of corneal epithelial cells, improvement of dry eye, or inhibition of inflammation.

[5] A metabolic syndrome comprising the composition according to any one of [1] to [4], wherein the metabolic syndrome includes oxidative stress reduction, lipid oxidation inhibition, prevention of ischemic diseases, improvement in metabolic syndrome, improvement in glucose tolerance, The present invention relates to a food composition for improving the protection of a disorder accompanied by an inflammatory reaction, a carcinogenesis inhibition, a reduction in systemic immunity, an inhibition of vascular endothelial dysfunction, an arteriosclerosis inhibition, a dementia prevention, a brain function improvement, a corneal epithelial cell protection,

[6] A food comprising the composition according to any one of [1] to [4] above.

[7] Formula I:

Wherein R is hydrogen, an erthioyl group, a glutathionyl group, or a systeinyl group,

The present invention relates to a method for the prevention of metabolic syndrome including prevention of oxidative stress, prevention of oxidative lipid, prevention of ischemic diseases, improvement of metabolic syndrome, improvement of impaired glucose tolerance, prevention of metabolic syndrome including metabolic syndrome, A pharmaceutical composition for suppressing the reduction of systemic immunity, inhibiting vascular endothelial dysfunction, inhibiting arteriosclerosis, preventing dementia, improving brain function, protecting corneal epithelial cells, improving dry eye or suppressing inflammation.

[8] Organic selenium compound represented by formula II:

The pharmaceutical composition according to the above [7], wherein the pharmaceutical composition is 3- (2-hydrocinol-1H-imidazol-5-yl) -2- (trimethylammonio) propanoate.

[9] Formula III:

As an active ingredient, an effective amount of an organic selenium compound represented by the following formula (1): (1) an oxidative stress reduction, a lipid oxidation inhibition, an ischemic disease prevention, a metabolic syndrome improvement, an impaired glucose tolerance, A pharmaceutical composition for suppressing the reduction of systemic immunity, inhibiting vascular endothelial dysfunction, inhibiting arteriosclerosis, preventing dementia, improving brain function, protecting corneal epithelial cells, improving dry eye or suppressing inflammation.

[10] Formula IV:

The present invention relates to a method for the prevention of metabolic syndrome including prevention of oxidative stress, prevention of oxidative lipid, prevention of ischemic diseases, improvement of metabolic syndrome, improvement of impaired glucose tolerance, prevention of metabolic syndrome including metabolic syndrome, A pharmaceutical composition for suppressing the reduction of systemic immunity, inhibiting vascular endothelial dysfunction, inhibiting arteriosclerosis, preventing dementia, improving brain function, protecting corneal epithelial cells, improving dry eye or suppressing inflammation.

The organic selenium compound of the present invention has been known to have an antioxidant ability that suppresses the generation of peroxides and radicals as an antioxidant based on the radical scavenging activity in the prior art. However, the demonstration of effectiveness of using a mammalian organism has not been disclosed until now. The anti-cancer and anti-arteriosclerosis are demonstrated by the efficacy test by administration to a human cell or a living body, and it can be used for new use such as prevention and improvement of diseases caused by lifestyle diseases and aging.

1 shows the chromatogram (A) of selenium 82 as a selenonane standard product and the chromatogram (B) of selenonane-containing fish extract obtained in Example 1. Fig.
Fig. 2 shows the effect of cerebral ischemia reperfusion by measuring spontaneous momentum after ischemic stress on sand rats and evaluating the effect of the selenium-containing composition (selenonine-containing fish extract) of the test substance in this model Fig.
Fig. 3 shows the effect of cerebral ischemia reperfusion (day 7) after loading cerebral ischemia-reperfusion in sand rats and Y-maze test. The selenium-containing composition of the test substance in this model ). ≪ / RTI >
Fig. 4 shows the effect of cerebral ischemia reperfusion (day 14) after loading cerebral ischemia-reperfusion in sand rats, followed by Y-maze test, and the effect of selenium-containing fish extract ). ≪ / RTI >
FIG. 5 is a diagram for explaining an improvement effect of the metabolic syndrome caused by ingestion of a selenium-containing composition in a mouse with glucose metabolism abnormality.
Fig. 6 is a diagram for explaining an improvement effect from injury accompanying metabolic syndrome caused by ingestion of a selenium-containing composition in a mouse with glucose metabolism abnormality.
FIG. 7 is a diagram for explaining the effect of suppressing the anticancer and systemic immune function depression due to the ingestion of the selenium-containing composition using the colon cancer priming model.
8 is a diagram for explaining the effect of suppressing the decrease of systemic immunity by consumption of a selenium-containing composition using a colon cancer priming model.
FIG. 9 is a diagram for explaining the effect of preventing recurrence of cancer by ingesting a selenium-containing composition based on inhibition of Treg cells.
FIG. 10 is a diagram for explaining the effect of inhibiting the promotion of atherosclerosis by ingestion of a selenium-containing composition based on inhibition of migration (smoothness) of smooth muscle cells.
Fig. 11 is a diagram for explaining the effect of ingestion of a selenium-containing composition for defending cells from the toxicity of amyloid beta.
Fig. 12 is a diagram for examining the change in viable count after drying stress due to consumption of the selenium-containing composition. Fig.
Fig. 13 is a diagram for examining changes in mucin gene expression by ingestion of a selenium-containing composition.
Fig. 14 is a view for examining the effect of inhibiting NFκ-B activation by ingestion of a selenium-containing composition.

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

(1) Organic selenium compound

The selenium-containing composition of the present invention can be obtained according to the method described in Japanese Patent No. 5669056, for example. Unless otherwise specified in the present specification, the "organic selenium compound" includes a selenonine monomer represented by the formula (II) and a tautomer represented by the formula (III), a selenoylphosphine dimer represented by the formula (IV) Quot; selenium-containing " selenine-containing chemical plant, and a " selenium-containing composition " containing these compounds. Here, the organic selenium compound can be obtained by obtaining a selenium concentrate by extracting a biological sample such as fish with an aqueous solvent or an organic solvent, and then separating and purifying it by chromatography. Here, the term " selenium concentrate " refers to a selenium concentrate containing the organic selenium compound (to be described later) represented by the formulas I to IV of the present invention. The sample is extracted with an organic solvent or water and then concentrated using a rotary evaporator And the like. In the case of a selenium concentrate in the form of a solution, it is preferable that the selenium compound of the present invention contains not less than 5 / / mL of the organic selenium compound of the present invention, more preferably dried powder dried by concentration under reduced pressure.

The organic selenium compounds to be subjected to the present invention are limited to the compounds of the formulas I to IV. Formula I:

, And the substituent R is an "organic selenium compound" bonded to a thiol compound presumably produced in vivo, such as hydrogen, erthioneone, glutathione, cysteine, acetylcysteine, homocysteine, methyl mercury, Selenium compound " also include metal or polymer materials bound through the selenium group.

In one embodiment, the organic selenium compound of the present invention has formula II:

Yl) -2- (trimethylammonio) propanoate represented by the formula (I) and the formula III:

3- (2-selenoxo-2,3-dihydro-1H-imidazol-4-yl) -2- (trimethylammonio) propanoate. As described above, the organic selenium compounds represented by formula II and formula III are all monomers of organic selenium compounds. The organic selenium compound of formula II has a molecular structure in which a seleno group is bonded to the carbon atom at the second position of the imidazole ring and the selenol group forms a tautomer in equilibrium with the selenoketone group, Depending on the conditions, it has two chemical forms: a selenol type (formula II) and a selenoketone type (formula III). The oxidized dimer is easily formed in the presence of a non-polar solvent in which the equilibrium tends to equilibrate with the selenol isomer. On the other hand, in the polar solvent, the chemical equilibrium is biased by the selenoketone isomer and exists mainly as a monomer.

In the organic selenium compound of the present invention,

(3, 3 '- (2,2'-dicellandiylbis (1H-imidazol-5,2-diyl)) bis (2- (trimethylammonio) propanoate) represented by the formula Includes 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 of the imidazole ring as a basic unit and the compound forms a diselenide through a seleno group.

According to the present invention, the content of selenium in the selenium-containing composition can be measured by a fluorescence method using 2,3-diaminonaphthalene (DAN) (for example, see JH Watkinson, Anal. Chem., 38, 92-97 (1966) (See, for example, H. Ge, et al., Anal. Commun., 33, 279-281 (1996)) using an HPLC-ICP-MS method using GPC columns.

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 (" selenonine "). The composition containing selenonine of the present invention may be in the form of tablets, pastes, or tablets containing 0.1 to 100 占 퐂 of selenone concentrate, dried or solidified selenone or selenonine as selenium, It may be a shape of a drink. When it is used as a food such as a supplement or a drink, it is preferable to form it into a shape suitable for taking the necessary amount of selenium per day. Other food materials, sweeteners, flavors, excipients, thickeners, preservatives, and the like.

As a typical example of the organic selenium compound of the present invention, raw materials derived from selenonin include, for example, tuna (bluefin tuna, southern bluefin tuna, yellowfin tuna, tuna, wing tuna), marlin (swordfish, Mackerel, mackerel, mackerel, mackerel, mackerel, mackerel, mackerel, striped mackerel), defense, mackerel, sardines (sardines) , Anchovy), marine mammals including cichlids (Japanese whales), and yeast (Schizosaccharomyces pombe) ("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. (7): e105177 Cham ).

(2) Selenium-containing composition

The present invention provides a selenium-containing composition containing the above-described organic selenium compound as an active ingredient. The content of the organic selenium compound contained in the composition of the present invention is within a range of 0.000001 to 99% by weight based on the total weight of the composition. Those skilled in the art can appropriately adjust the content of the organic selenium compound to obtain a desired effect. When the selenium-containing composition is used as an active ingredient, the selenium-containing composition may contain 0.1 to 100 μg of selenium as described above. The raw material for the composition is not particularly limited as long as it contains selenonine. For example, the microorganism such as fish, marine mammal and yeast may be used as a raw material for the composition, such as mycelium, plant, seaweed, algae, Chemical synthesis can also be a raw material.

The organic selenium compound of the present invention may contain other components in addition to the organic selenium compound as an effective ingredient, depending on the purpose. Other components that may be contained in the selenium-containing composition of the present invention include, but are not limited to, water; Alcohol; Processed meat products; Rice, wheat, corn, potato, sweet potato, soybean, kelp, seaweed, mugwort, and powder thereof; Sugars such as starch, starch syrup, lactose, glucose, fructose, sucrose and mannitol; Proteins such as fish meal powder, egg whites and dried egg whites (whole eggs), fish eggs, fish protein, plant protein, collagen, myosin and gluten; General food additives such as spices, sweeteners, edible oils and vitamins; Surfactants; Excipients; flash; Preservative; Coating aids; Lactose; dextrin; cornstarch; Sorbitol; Crystalline cellulose; Polyvinylpyrrolidone; Oil; Moisturizer; Thickener; antiseptic; Spices; And combinations thereof. The composition of the present invention may also contain other medicines if necessary. The content of these other components and / or other agents is not particularly limited and may be suitably selected by those skilled in the art.

(3) foods, food compositions and medicinal compositions containing selenium-containing compositions

According to the present invention, a food, a food composition and a medicinal composition containing the composition of the present invention are provided. When the composition of the present invention is used as a food and a food composition, it may contain a carrier or other additive which is acceptable as a food according to a desired product form. Examples of such carriers and additives include excipients, binders, perfumes, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, disintegrating agents, lubricants, preservatives and the like. The pharmaceutical composition of the present invention may be in the form of a solid preparation such as a food, a food additive, a tablet, a powder, a granule, a granule, a capsule, a pill or a sustained release, a liquid preparation such as a solution, a suspension, . These can be formulated or commercialized together with a carrier or an additive agent, if necessary, according to a technique commonly used in the art.

(4) Usage

The selenium-containing composition containing the selenium compound of the present invention as an effective ingredient of the selenium compound of the present invention and the food and food composition containing the composition of the present invention are useful as an antioxidative stress reducing agent, a lipid oxidation inhibitor, an ischemic disease prevention agent, a metabolic syndrome improving agent, , Prevention of metabolic syndrome including lipid metabolism, prevention of cancer, suppression of systemic immunity, inhibition of vascular endothelial dysfunction, prevention of arteriosclerosis, prevention of dementia, improvement of brain function, protection of corneal epithelium, improvement of dry eye And inflammation inhibition are expected. In the aspect of use as food, though not limited, it may be in the form of a health food, a functional food, a specific health food, a nutritional functional food, a nutritional supplement food, a functional food, a drink or the like. As used herein, the term " improvement " refers to an improvement or prevention of a disease, a symptom or a condition, an prevention or delay of a disease, a symptom or a deterioration of the condition, or a reversal, prevention or delay of progression of a disease, symptom or condition. "Abatement" refers to a delay in the appearance of a disease, a symptom or a condition, a decrease in frequency, and a decrease in severity, and may be used in agreement with "improvement". In addition, " inhibition " means to completely or partially inhibit a disease, symptom or condition. In the present specification, the above-mentioned " improvement ", " reduction ", and " inhibition " On the other hand, " prevention " refers to preventing or delaying the development of a disease or symptom in an individual, or reducing the risk of developing a disease or symptom of an individual. The term " protection " means prevention of development of various clinical symptoms, and may be understood to include " prevention ".

In the selenium-containing composition of the present invention, in order to obtain the above-described desired effect, the organic selenium compound as an active ingredient is added in an amount of 0.1 ng to 10 mg, more preferably 1 ng to 5 mg, More preferably from 3 ng to 1 mg, still more preferably from 5 ng to 500 μg, even more preferably from 5 ng to 100 μg, and most preferably from 10 ng to 50 mg, Mu] g. More specifically, it is preferably administered or ingested from 2 ng to 2550 μg per day, more preferably from 5 ng to 47 μg, and still more preferably from 10 ng to 14.5 μg per day. In the present invention, such an effective ingredient may be administered once or several times a day, and then administered or ingested in the form of the compound or in a desired form such as a food, a composition, a medicament, a food or the like .

a. Prevention of ischemic diseases and reduction of oxidative stress

In ischemia, it is known that a large amount of reactive oxygen is generated during reperfusion of blood, resulting in a disorder. The ingestion of the selenium-containing composition of the present invention enhances the bio-antioxidant action, thereby alleviating the oxidative stress in the brain and preventing ischemic brain diseases. The effect of preventing ischemic diseases and the effect of reducing oxidative stress can be confirmed, for example, by administering a selenium-containing composition, followed by a cerebral ischemia reperfusion model test using a sand mouse, to alleviate ischemic reperfusion injury. More specifically, sand rats are administered a selenium-containing composition (for example, selenenoine-containing fish extract) for 2 weeks, and the blood flow of the bilateral total carotid artery is stopped for 5 minutes before reperfusion. Thereafter, the administration of the organic selenium compound is continued for 2 weeks, and the spontaneous momentum and the work memory ability can be examined and evaluated (see Example 2 described later). In addition, the brain and the heart may be removed after slaughter to confirm the ischemic site biochemically. The selenium-containing composition of the present invention can be used as an effective ingredient of food materials and compositions that protect against defects due to alleviation of oxidative stress caused by oxygen deficiency, ischemic heart disease, cerebral infarction and the like.

b. Improvement of protection from metabolic syndrome and its accompanying disorders

Alcohol consumption, viruses, etc., as well as non-alcoholic hepatitis patients are increasing recently. The ingestion of the selenium-containing composition of the present invention can prevent the suppression of lipid peroxidation (oxidized LDL), nonalcoholic hepatitis, fatty liver, abnormal glucose tolerance, metabolic syndrome and accompanying diabetes mellitus. As an animal model, a non-alcoholic hepatitis model mouse based on diabetes mellitus was prepared by administering streptozotocin and a high fat diet, and the selenium-containing composition of the present invention was mixed with the diet to give an oxidative stress and / It is possible to confirm the preventive effect against liver disorder. For example, by taking the selenone-containing fish extract of the present invention, which is a selenium-containing composition of the present invention, and a high-fat diet, the liver is taken out after 4 weeks and the hepatitis score is measured to reduce the oxidative stress caused by the selenium- (See Example 3 to be described later), the effect of reducing fat accumulation in the liver, the development of hepatitis and metabolic syndrome, and the improvement from the disorder.

c. Prevention effect of cancer prevention and suppression of decrease of systemic immunity

The ingestion of the selenium-containing composition of the present invention can enhance the inhibitory effect on cancer proliferation, metastasis and cancer, and the effect of suppressing the decrease in systemic immunity. By using an animal model, it is possible to confirm a carcinogenesis prevention effect and an effect of suppressing a decrease in systemic immunity. The proliferation inhibitory effect of cancer cells can be confirmed by implanting cancer cells into a mouse and measuring the decrease in tumor volume when the feed containing the selenium-containing composition of the present invention (for example, a selenium concentration of 3 mg / kg) is administered .

The effect of inhibiting mutagenicity by the selenium-containing composition of the present invention can be confirmed by a return mutation test using a bacterium. In addition, under a condition closer to a living body, mammalian-derived cells (for example, Bhas42 cells) can be used to confirm the effect of inhibiting the transformation by the selenium-containing composition of the present invention. This method is a test method used for the detection of carcinogens established at the Food and Drug Center, a general foundation of Japan, and can also detect the initiation and promoting activities of carcinogenesis. One day after seeding the cells, the transforming inducer 3-methylcolanthrene is added to the medium and treated for 3 days. The cells were exchanged with a medium supplemented with selenonine-containing fish extract, cultured until day 21 after cell seeding, and then the transformants (transgenic plants) were counted. Since the effect can be confirmed at each stage of cancer initiation, promotion, and progression, it can be evaluated whether it is usable as a food material having cancer prevention effect (see Example 4 described later).

In addition, the selenium-containing composition of the present invention may be administered to a gangrenous (膽 cancer) mouse to confirm the effect of improving the whole-body immunity. Regarding the suppression of systemic immunomodulation lowering, the cancer-inhibiting effect of the selenium-containing composition can be verified by analyzing a control T (Treg) cell having an immunosuppressive function and promoting tumor growth (see Example 4 described later) . The ingestion of the selenium-containing composition of the present invention improves the immunological ability of the subject to be treated, thereby inhibiting the growth of cancer or preventing cancer recurrence.

d. Atherosclerosis-inhibiting effect

Oxidized LDL is thought to be a part of the atherosclerotic development and it has been reported that smooth muscle cells are migrated by oxidized LDL. It has been found that the selenium-containing composition of the present invention has an effect of inhibiting arteriosclerosis, which inhibits dehiscence of smooth muscle cells and migration to the lumen of the blood vessels, which cause stenosis at the time of development of arteriosclerosis. The selenium-containing composition of the present invention can be administered to smooth muscle cells to inhibit the smooth muscle cell migration and to inhibit arteriosclerosis. For example, human aortic smooth muscle cells are cultured in a medium supplemented with a selenium-containing composition, and the number of migrated cells can be measured to evaluate whether or not the cells can be used as a food material having an arteriosclerosis-preventing effect (see Example 5 described later) . According to the present invention, it is possible to inhibit the smoothness of smooth muscle cells and to suppress the promotion of atherosclerosis, for example. In addition, vascular endothelial disorders associated with arteriosclerosis can be improved.

e. Brain function improvement effect

As risk factors for Alzheimer's disease, aging, genetic predisposition, lifestyle diseases (diabetes, etc.) are known. It has been found that the selenium-containing composition of the present invention has an effect of improving brain function to prevent the development of dementia by alleviating the generation of active oxygen species such as diabetes. In the present specification, " dementia " means Alzheimer's dementia, rubio dyskinesia, cerebrovascular dementia, frontotemporal dementia, and the like. The term " brain function " means a memory function (e.g., working memory, short term memory), emotion, language function, cognitive function, exercise function, learning function, attention function, and the like. Thus, the brain function can suppress the functional deterioration by protecting the ischemic reperfusion injury and the cranial nerve in various aspects. In addition, it is known that the risk of depression is increased due to diabetes, and it is possible to prevent not only dementia but also depression by protecting against brain dysfunction caused by diabetic disorder. As for the brain function improving effect as described above, for example, an Alzheimer's disease model mouse can be produced by administration of streptozotocin to confirm the effect of reducing the Alzheimer's progress by administration of the selenium-containing composition of the present invention (for example, selenonine-containing fish extract) have. It has been known that administration of streptozotocin directly into the brain increases oxidative stress in the brain, resulting in the accumulation of amyloid beta and tau protein, resulting in conditions such as Alzheimer's disease. For the model preparation, streptozotocin is administered after one week of ingesting the selenium-containing composition of the present invention. Thereafter, the selenium-containing composition is ingested for two weeks, and evaluation can be made by examining working memory and brain tissue. In addition, it is possible to biochemically confirm the amyloid-β accumulation-inhibiting action by a cell-based test. It is expected that the consumption of the selenium-containing composition and the food containing the selenium-containing composition having the effect of preventing and reducing the brain function deterioration has a brain function-protecting effect not only for the patient but also for the healthy elderly person (see Example 6 described later).

f. Dry eye improvement effect

By ingesting the selenium-containing composition, the drying and oxidative stress of the corneal epithelial cells are alleviated and protected, and a dry eye prevention effect can be obtained. Dry eye patients are said to be about 8 million to 22 million people in Japan. It has been reported that inflammation or persistent oxidative stress exacerbates the dry eye, resulting in the formation of mitochondria in mucin-less or dry-model mice (Kawai, M., et al., Sci. Rep., Vol. 2013). Human immortalized corneal epithelial cells can be used for evaluation by measuring the production of mucins and the like by culturing them in a medium supplemented with an organic selenium compound (for example, selenonine-containing fish extract). Although dry eye is not a major disease, it tends to be overlooked. However, improvement of dry eye is very important for improvement of QOL of patient and it can be evaluated whether or not it can be used as a raw material for improving dry eye (see Example 7 described later). By using the selenium-containing composition of the present invention, dry stress of the corneal cells can be protected, and dry eye can be prevented and improved.

g. Inflammation inhibitory effect

The ingestion of the selenium-containing composition of the present invention can prevent erythema, swelling, fever, pain and dysfunction due to inflammation and prevent rheumatoid arthritis, hemolytic anemia and myasthenia gravis caused by inflammation. In normal persons, the immune regulating mechanism functions in a well-balanced manner. However, when an abnormality occurs in the host's immunoregulatory mechanism due to any cause, a magnetic antibody recognizing its own biomolecule as an antigen is produced and causes various immune responses, (Fibrosis) disorder, dysfunction is occurring. Examples of such autoimmune diseases include rheumatoid arthritis, hemolytic anemia, and severe myasthenia gravis. Immunosuppressive agents such as azathioprine, cyclophosphamide and vincristine are used as therapeutic agents for such autoimmune diseases. However, it is known that side effects due to the administration of these agents also spread over many aspects. However, most of the exogenous inflammatory drugs applied directly to the affected area were steroids, and their use was limited due to their side effects. The organic selenium compound of the present invention can be used as a food, has no side effects, and is expected to suppress various inflammatory reactions. An object of the inflammation-suppressing effect by the ingestion of the selenium-containing composition of the present invention is an effect caused by NFκ-B activation, and is useful as an anti-inflammatory agent for preventing or treating inflammation, for example, an allergic disease, an anti- Spondylitis, celiac disease (see Example 8 to be described later).

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

Example

Example 1: Preparation of organic selenium compounds

As the organic selenium compound, an extract containing selenonine was obtained from the tuna fish meat bleaching water according to the method described in Japanese Patent No. 5669056 (Patent Document 1), and the extract was concentrated to Brix 55 to obtain a selenonine-containing fish An extract was obtained.

Analysis of selenonine content

The selenonine content of the selenonane-containing fish extract (Brix55) obtained in Example 1 was calculated as the selenium equivalent (S Se / g) based on the measurement value obtained by the following LC-ICPMS.

(1) Device configuration

Separation column: Ultrahydrogel 120 (Waters)

LC pump: Pu712 (GL Science Inc.)

Sample Injector: 9725i (Rheodyne)

ICPMS: ELAN DRC II (Perkin-Elmer)

(2) LC measurement conditions

Mobile layer: 0.1% IGEPAL-CA63, 0.1 M ammonium acetate (pH 5.3)

Flow rate: 1.0 mL / min

Sample injection amount: 0.01 mL

(3) ICP-MS measurement conditions

Measuring element: Se

Mass: 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

Reaction gas (Ar, L / min): 0

Pulse stage voltage (eV): 1050

RPq: 2.5

Staying time (sec / amu): 0.4

(4) Preparation of samples

The seleneno-containing fish extract obtained in Example 1 was mixed with 19-fold volume (380 μL) of ultra-pure water (Milli-Q Element, Millipore Co., Ltd.) for 20 μL and centrifuged at 12,000 rpm for 10 minutes. - Used for ICPMS analysis.

(5) Preparation of selenone standard

The selenone standard was obtained from the swordfish sphaerothecae in the following manner:

(5-1) Frozen swordfish fillets were thawed overnight at 5 ° C, and the collected syringes were cut out and used.

(5-2) 0.1 g of cysteine hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 100 g of hemostatic muscle, and a Polytron homogenizer (PT MR 6000, manufactured by Wako Pure Chemical Industries, Ltd.) KINEMATICA) at 6800 rpm for 15 seconds while cooling with ice water.

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

(5-4) To concentrated solution 1, a mixed solution of acetonitrile: tetrahydrofuran (Wako Pure Chemical Industries, Ltd.) (1: 1, v / v) was added and the concentrate was separated into two layers.

(5-5) The acetonitrile layer was separated from the separated two layers, and the concentrated liquid 2 was obtained by concentrating the liquid with a rotary evaporator under reduced pressure.

(5-6) 5 ml of cooling water was added to Concentrated Liquid 2, and the solution was supplied to an Atlantis (registered trademark) dC18300 Angstrom column (19x150 mm, Nippon Waters) equilibrated with 0.1% acetic acid to obtain 0-50% linear acetonitrile Under the gradient, fractions 1 from 40 ml to 50 ml of the eluate were recovered.

(5-7) Fraction 1 was concentrated under reduced pressure on a rotary evaporator to obtain Concentrated Liquid 3.

(5-8) Concentrate 3 was supplied to an Ultrahydrogel (TM) 120 column (7.8 x 300 mm, Nippon Waters Co.) equilibrated with 0.1% acetic acid 30% acetonitrile and a solution of selenonine Were collected and a fraction 2 was obtained.

(5-9) Fraction 2 was passed through an Ultrahydrogel (registered trademark) 120 column (7.8 × 300 mm, Nippon Waters Corporation) equilibrated with 0.1% acetic acid, and fractions containing selenonine were collected and concentrated under reduced pressure on a rotary evaporator And concentrated to obtain a selenone standard.

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

The selenonane standards obtained in the above (5-1) to (5-9) include the compounds represented by the formulas (1) to (4), and the compounds represented by the formula (2) (Yamagishi Yumiko, 2012)). [0004] In recent years,

(6) Identification and quantification of selenonine

Selenium 82 was measured by using the LC-ICPMS apparatus described in (1) to (3) with respect to the sample of (4) and the selenone standard, and the chromatogram was obtained, and the identity and content of selenonine were determined using these .

(6-1) A peak is detected near the elution time of 10.3 minutes in the selenone standard product. In addition, the selenonane standard in which the signal of the tiny selenium compound was detected was regarded as the peak area corresponding to the total amount of selenium injected, which was measured by the fluorescence method described later, by measuring the integral value of all selenium signals of 6-11 minutes.

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

(6-3) Formula (5) below:

X = (A / B) x (1000 / C) x D / 1000 (5)

(here,

X: Content of selenonine in the sample (占 퐂 Se / g)

A: Peak area of 10.3 minutes of sample

B: detection area per injection selenium weight of the selenone standard (/ ngSe)

C: The micro L amount of the injected sample in the LC-ICPMS (now C = 5)

D: Dilution ratio by ultrapure water when the sample is prepared (D = 20 at this time).

(6-4) 10 μL of selenonane standard was injected into the LC-ICPMS device, and the integral value of all selenium signals for 6-11 minutes was measured and the integral value was divided by the amount of injected selenium (this time, 4.76 ng) Was determined as the detection area per weight of selenium (coefficient C in the equation (5)

(6-5) Measurement result

1 shows the chromatogram (A) of selenium 82 of the selenonane standard product and the chromatogram (B) of the selenonane-containing fish extract obtained in Example 1.

(6-6) In the chromatogram (B), a peak (arrow) corresponding to the selenonine dimer (Formula IV) was observed as a major selenium compound at a retention time of 10.6 minutes.

Therefore, most of the selenium compounds contained in the seleneno-containing fish extract obtained in Example 1 were found to be the main component of the compound represented by the formula IV. With respect to the seleneno-containing fish extract obtained in Example 1, the calculated selenonine content was 32.5 mgSe / L.

(7) Total selenium content

The selenenoyl-containing fish extract obtained in Example 1 and the selenone standard obtained in (5-1) to (5-9) were measured by using a fluorometric determination of selenium in nanograms in biological materials using 2,3-diaminonaphthalene. R By wet decomposition according to the method described in Hasunuma, T. Ogawa, Y. Kawanishi, Analytical biochemistry (1982), 126: 242-245) to convert all of the selenium compounds into inorganic form, Was measured by measuring the fluorescence of 4,5-benzopia selenol (Se-DAN) produced by reaction with minonaphthalene (DAN) and complex formation reaction with Se (IV). And calculated as selenium weight (mg / kg) per sample weight based on the obtained measurement values. Specifically, the measurement was carried out under the following conditions.

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

(7-2) After water-cooling the sample obtained in (7-1), 0.25 mL of 6 M hydrochloric acid was added and the mixture was heated in a water bath at 100 ° C for 30 minutes. After water-cooling, 0.12 M disodium ethylenediaminetetraacetate 0.25 mL And the pH was adjusted to 1.0 to 1.5 using a 6 M aqueous solution of sodium hydroxide.

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

(7-4) A calibration curve was prepared using a test tube containing 5, 10, 50, and 100 μL of the operation blank and a 1 mg / L selenium standard solution in parallel with the sample, and the total selenium content in the sample was calculated.

The selenium-containing fish extract obtained in Example 1 had a total selenium content of 95.3 mg / kg.

Example 2: Examination of reduction effect of ischemia reperfusion injury

After the cerebral ischemia reperfusion was applied to the sand rats, spontaneous momentum measurement and Y-maze test were performed to examine the effect of cerebral ischemia reperfusion and the effect of the test substance (selenonine-containing fish extract) in this model did.

1. Test system

Seven-week-old sand rats (MON / Jms / Gbs Slc, male) were purchased from Nisshin SL, Inc. for 5 days, and their weights were measured and the groups were divided. During the test period, feed (CRF-1, Oriental Kogyo Co., Ltd.) and water were freely consumed. The test group was divided into the following six groups.

On Day 14, the day of administration of the test substance was set to Day 0, and under the isoflurane anesthesia on the day 14, the entire carotid artery was threaded on the day before ischemia to seal the incision. On the next day, to keep the body temperature at 37 ° C, the incision was cut on a hot plate set at 40 ° C ± 3 ° C, and the both common carotid arteries were pulled and the arteries were hemostatically clamped with Klemme for 5 minutes. Thereafter, the forceps were removed and reperfusion was performed, and the incision was sealed. In the normal group, the bilateral total carotid artery was exposed under isoflurane anesthesia and the skin incision was sutured without treatment. For the group of administration of the test substance, 6.2 mL / kg of the test substance was forcedly administered using a polypropylene disposable syringe and gastric sonde daily during the test period. Normal and control groups received water in the same manner as 6.2 mL / Kg. Compulsory dosing was done for 22 days or 29 days at a frequency of once a day. When ischemia reperfusion is performed, behavioral abnormality (spontaneous motor hyperactivity) or learning memory disturbance occurs due to somatic cell death. Spontaneous momentum measurement and Y maze test were performed to evaluate the protective effect of the brain disorder caused by ischemia reperfusion.

(1) Spontaneous momentum measurement

The spontaneous momentum was measured for each hour on Day 6, 13, 15, 17, 19, 21, and 27 (Day 2 was only Second Kur). For the measurement, a multi-channel spontaneous momentum measurement system, Spamex (Muromachi Kikai K.K.) was used.

(2) Y maze test

The Y-shaped maze test evaluated memory learning behaviors by putting animals on Day 21 (1st Cool) and Day 28 (2nd Cool) and letting them behave freely for 6 minutes and measuring the rate of selecting new arms.

(3) Measurement of selenonine

At the end of the first cool, blood was euthanized by collecting whole blood from the abdominal aorta of the animal into a syringe containing heparin under 2.5% isoflurane inhalation anesthesia from the surviving specimens of groups 1, 2 and 3. The collected blood was centrifuged (3000 rpm, 10 minutes, 4 ° C) to collect plasma and blood clots. The content of selenonine in blood clots was analyzed in accordance with the method of Example 1.

2. Results

1 day after the ischemia reperfusion treatment, the spontaneous momentum was increased in the control group 2 (control 22 days group in Fig. 2) and 5 group (control 29 days group in Fig. 2) 2 was significantly inhibited in Group 3 (group of "22-day administration of selenone-containing fish extract" in FIG. 2) and group 6 ("29 days of administration of selenone-containing fish extract" in FIG. 2) ). On the other hand, when the Y maze test was conducted after 7 days and 14 days after the treatment, the correct answer rate decreased in the control group, but the decrease was significantly inhibited in the selenone-containing fish extract-treated group, ), It was confirmed that the test substance had an effect of protecting from brain damage caused by ischemic reperfusion (FIGS. 3 and 4).

There was no significant difference in selenium protein content between the groups 1, 2 and 3, but selenonine content was less than the detection limit (0.01 ㎍ / g) in group 1 and 2 In group 3, 0.95 ± 0.24 ㎍ / g was detected per selenium, and selenonine content was significantly increased (p <0.05). From these results, it was revealed that selenonine was accumulated in the blood cells of the sand rats by the administration of the test substance for 22 days.

Example 3: Examination of the effect of improving the protection from the metabolic syndrome and the following disorders

(1) Improvement effect of metabolic syndrome

By the administration of streptozotocin, the mice that had hyperglycemia, like type I diabetes, were fed a high fat diet. The NAFLD activity score, which is a method capable of comprehensive evaluation of liver fat accumulation and the like after a predetermined period, was used to verify the non-alcoholic hepatitis inhibitory effect by administration of selenonine-containing fish extract.

More specifically, 10 females of an SPF mouse (C57BL / 6J, Nippon SL) were purchased on the 14th day of pregnancy and cultivated. Whole animals were spontaneously delivered and male mice born were used to make model animals. In addition, culling was made at 4 weeks of age.

Observation of Born Births: For mice in which births were observed at 8:00 births, the day of observation was set as the day before the day of observation, and for mice in which births were observed at 20:00 as a birth, , And was calculated from day 0 of birth. Streptozotocin (Sigma Aldrich Japan Co., Ltd.) was injected into male mice at 2 days of age with a physiological saline solution (Japanese Pharmacopoeia, Otsuka Seiyaku Plant, Ltd.) at a concentration of 10 mg / 200 [mu] g / dose). After that, I had my mother give me nursing until weeping. At 28 ± 2 days of age, the mice were weaned, and then fed with high fat diet 32 (radiation sterilization, Nippon Clea Co., Ltd.).

From the day of admission to feeding, all of the mothers were free to ingest the solid feed CE-2 (radiation sterilization, Nippon Clea Co., Ltd.). After the weaning, high fat Diet 32 (radiation sterilization) (Nippon Clea Co., Ltd.) was freely taken for the animals to which streptozotocin was administered.

One dose was a selenone-containing fish extract at 15 mg / kg body weight. The dose was adjusted to 10 mL / kg body weight, and oral oral administration was performed using oral sonde. The control group was administered in the same manner at a dose of 10 mL / kg. The dose of each individual was calculated based on the weight of the morning (unit: 0.01 mL). The period was from 28 days to once a day from 5 weeks of age. After the completion of the administration, the liver was removed by abdominal aortic dissection, the liver was extracted, the outer left lobe was divided into six equal parts, and the two pieces were stained with hematoxylin-eosin to calculate the NAFLD activity score.

NAFLD activity scores are scored for hepatocyte localization, inflammation of liver parenchyma, and hepatocyte disturbance (ballooning degeneration), and are used for pathologic diagnosis of nonalcoholic hepatitis. In the control group, inflammatory cell infiltration was observed including infiltration of lipids (lipid droplets), ballooning of hepatocytes, large droplets and small droplets, lymphocytes and neutrophils. The NAFLD activity score was significantly lower in the selenonane-containing fish extract-administered group than in the control group. Selenonine was suggested to prevent and improve fat accumulation due to metabolic syndrome caused by metabolic syndrome, and the prevention and improvement effect of metabolic syndrome was considered (Fig. 5, Table 3).

(2) Improvement effect from disability accompanying metabolic syndrome

The icv-STZ-induced Alzheimer's dementia model is a model animal of Alzheimer's type dementia with the background of diabetes mellitus caused by impairing the insulin signal in the brain. Therefore, this model is useful for examining the therapeutic effect on dementia with sporadic Alzheimer's disease. In this example, icv-STZ induced Alzheimer's type dementia model mice were injected with the model induction 7 days before the start of the model, with the aim of examining the effects of selenonine-containing fish extract as a test substance on Alzheimer's dementia Forcible oral administration was performed for 21 days from the start to 14 days. Behavioral analysis was performed on the 13th day after the start of the model induction, and the animals were slaughtered on the 14th day after the start of the model induction and analyzed. The effect of selenonine-containing fish extract on Alzheimer type dementia in icv-STZ-induced Alzheimer's dementia model by comparison of solvent administration group (RO number, control administration group) with selenonane-containing fish extract administration group .

Streptozotocin (Sigma Aldrich Japan Co., Ltd.) was added to a physiological saline solution (Japanese pharmacopoeia, Otsuka Seiyaku Co., Ltd.) to a mouse (C57BL / 6J, 6 weeks old at the time of arrival, At a concentration of 20 mg / mL, in the lateral ventricle. An icv-STZ induced Alzheimer-type dementia model animal was prepared by administering 3 μL once per day to the left ventricle on the day of model induction and 3 μL on the second day after the start of model induction. On the day before the start of administration, 20 male mice were randomly divided into body weight-divided randomly so that the average body weight became uniform, The groups were divided into icv-STZ + control group and icv-STZ + selenonane-containing fish extract administration group (10 rats / group). The dose of the test substance was 6.2 mL / kg body weight, and oral oral administration was conducted once a day for 21 days using oral sonde. The control group was administered in the same manner with RO number of 6.2 mL / kg body weight. The dose of each individual was calculated based on the weight of the morning (unit: 0.01 mL).

After the end of the administration period, the spontaneous replacement behavior by the Y maze was analyzed. One day before the end of the administration, the mice were placed in the center of the maze and allowed to freely navigate for 5 minutes. After 5 minutes had elapsed, the mouse was returned to the home cage and the Y maze was disinfected with 50% ethanol, followed by behavioral testing of the next mouse. On the day of the end of dosing, two of the three arms were partitioned into walls and the mice were placed in the center of the maze. After the mouse entered the opened arm, the wall installed at the arm entrance of the remaining two directions was removed, and the user was allowed to freely search for 8 minutes. During this time, the total number of times the mouse entered the arm and the order in which it entered the arm were all recorded. After a lapse of 8 minutes, the mice were returned to the home cage, and the Y maze was disinfected with 50% ethanol, followed by the next mouse test. The replacement activity rate (%) was used as an indicator of SAB. The replacement behavior rate was calculated by the following equation (Fig. 5, Table 4).

Replacement rate = 100 × (the number of times the robot has entered the other arm continuously three times) / (the total number of times the robot has entered the arm) -2

As a result, the selenenoine-containing fish extract group showed a significantly higher replacement activity (P <0.05) than the control. It has been suggested that selenonine intake protects against brain dysfunction due to diabetes.

Example 4: Examination of anti-cancer effect or suppression effect of lowering of general immunity

(1) Verification of anticancer effect and suppression of systemic immunity lowering effect using colon cancer source model

The effect of selenenoine-containing fish extract on tumor growth and systemic immunity in cancer patients was examined using animal model animals. In each group, the ratio varied from 1.73 to 2.65 g per day. The intake of selenenoine-containing fish extract from selenenoine-containing fish extract feeds varied from 81.48 to 125.82 mg per day. As a result of using the colon cancer source chemotaxis model, there is a tendency that the growth of the primary tumor tumor is suppressed. Therefore, a significant decrease in the IL-10 acid productivity observed in the cancer cells is not observed in the selenonine-treated group, It seemed possible. In addition, regulatory T (Treg) cells were reduced by administration of selenonine, suggesting that the modulation of antitumor immunity by Treg cells may be improved. That is, it was suggested that selenonine has an effect of improving the systemic immunity of cancer patients and inhibiting tumor growth (FIGS. 7 and 8).

(R8758, manufactured by Sigma-Aldrich) containing 10% fetal bovine serum (171012, Nichirei Bioscience) in a Colon Tumor 26 (CT-26) strain (ATCC No. CRL-2638) . In addition, mouse non-cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 50 μM 2-mercaptoethanol (21417-52, Nacalai Tesque). Cells were cultured under conditions of 37 ° C, 5% CO ₂ , and 100% humidity using a water jacket incubator (APC-30D, Astec). Mice (BALB / c female, Charles River) were fed selenonine-containing feed or control feeds from 5 weeks to 8 weeks of age (see Table 5, below). After one week of purification (6 weeks of age), CT26 cells per mouse were implanted with 500,000 cells subcutaneously. Fourteen days after transplantation (8 weeks old), mice were euthanized, the tumors were harvested, and the tumor volume was measured. Tumor volume (㎣) was calculated by the formula (2) of width (mm) × length (mm) × 0.5. A subset of CD4-positive Foxp3-positive control T cells (Treg) in non-cells was extracted from the spleen using flow cytometry using a fluorescent staining cell analyzer (AccuriC6, BD bioscience) and FlowJo software (TreeStar) ). The concentration of IL-10 in the culture supernatant of non-cells was measured by ELISA.

Raw materials other than fish extracts of AIN-93M and selenonine-containing feed were purchased from Oriental Gobosha.

Controlled T (Treg) cells have immunosuppressive ability and promote tumor growth, but it is strongly suggested that selenonine-containing diet may inhibit Treg cells in dendritic mice. In addition, the IL-10 acid function which regulates antitumor activity by regulatory immunity was significantly reduced in dendritic mice, but not in the selenonine-containing diet group. Tumor growth was also inhibited. As a result, the immunological ability of the whole body was improved by selenonine, suggesting the effect of suppressing cancer growth. It was also thought that the inhibition of cancer recurrence by the inhibition of Treg cells (Fig. 9).

(2) Verification of the inhibitory effect of Bhas 42 cells on carcinogenesis

Transformation test using Bhas 42 cells was carried out to investigate the in vitro inhibition of carcinogenesis in selenonine, which is the main component of the selenonine purified product. In the capacity setting test, Bhas 42 cells were treated with selenonine purified products (0.31, 0.63, 1.3, 2.5, 5.0, 10 μM), and no cytotoxic effect was observed (data not shown). Thus, based on this and other experimental data, the concentration in the transformation test was set at 1 μM. In the transformation test, it was examined whether the number of transformants induced by 1 占 퐂 / mL of 3-methylclarenthrene (MCA) (Sigma-Aldrich) was reduced by 1 μM of selenonine purified product. That is, the transformation ratio (transformant number / well) in the MCA + ultrapure water group and the MCA + selenonine purified product group was compared. As a result, the respective transfection rates were 15.8 and 10.2, and a significant decrease in the number of transformants was recognized. From the above results, it was suggested that selenonine has a carcinostatic action in vitro.

Bhas 42 cells (cells in which the v-Ha-ras gene was introduced into mouse whole fetal-derived BALB / c 3T3 A31-1-1, Japan Food and Drug Safety Center, Japan) were detached using 0.25% trypsin, Lt; 2 &gt; cells / mL. 2 mL (4000 cells) of the cell suspension was dispensed into plates (6 wells / group) and cultured for 1 day. One day after sowing, the MCA preparation solution was added and treated for 3 days. Treatment of the selenonine purified product and RA (retinoic acid, Sigma-Aldrich) was carried out by changing the culture medium (2 mL per well) after 4 days, 7 days, and 11 days after sowing. After 14 days of sowing, it was exchanged with the fresh medium (treated for 10 days) and cultured for another 7 days. The plate for the transformation test was fixed with methanol after 21 days of sowing, and then stained with 5 vol% of the plating solution, and the transformant per well was counted.

The transformation rate (15.8 cells / well) of the MCA + selenonine purified product group was reduced to about 2/3 compared with that of the MCA + ultrapure water group (10.2 cells / well), showing a significant decrease. From these results, it was suggested that selenonine, which is a major component of the selenonine purified product, has a cancer-suppressing action.

Example 5: Examination of the effect of inhibiting arteriosclerosis

The effect of human aortic smooth muscle cells on cell migration of the test substance was examined. As a result, in the presence of collagen, the fish extract containing 2 mg / ml of selenonine and 100 nM of purified selenonine suppressed osmoticity.

AoSMC cells (aortic smooth muscle cells) (LONZA, CC-2571) were adjusted to the required number of cells with a dedicated medium (SmGM-2 Bullet Kit (LONZA, CC-3182)). AoSMC cells were cultured at 1 × 10 ⁵ cells / 0.1 mL / well using a cell migration assay kit (CytoSelect ^™ 96-well Cell Migration Assay (8 μm), Fluorometric, Cosmo Bio, CBA-106) A test medium containing the test substance (fish extract containing 2 mg / ml of selenonin or 100 nM purified selenonine) and 300 / / ml collagen peptide was added to the seeds of the transwells attached to the milk analysis kit And cultured for 48 hours. Thereafter, the cells were peeled and dissolved from the transwell membrane with CyQuant GR Dye attached to the cell migration assay kit, and the cells were immobilized.

The addition of purified selenone and selenonine - containing fish extract significantly inhibited cell viability. It was suggested that inhibition of smooth muscle cell metaplasia could inhibit the promotion of atherosclerosis (FIG. 10).

Example 6: Examination of brain function improvement effect

Amyloid beta protein, which is thought to be one of the causes of Alzheimer's disease, has neurotoxicity. The effect of preventing dementia can be expected by defending cells from the toxicity of amyloid beta. In this example, it was verified that the toxicity of amyloid beta can be protected by the test substance using PC-12 cells.

PC-12 cells (Ricken Bio Resource Center, RCB0009, Lot No. 52, 53, 54) were inoculated into 395 ml of DMEM medium (DMEM medium (Nacalai Tesque, Cat. No. 08456-65) supplemented with 10% , penicillin-streptomycin solution using a 5 ml and FBS (. Biowest, Cat No. S1820 , Lot No. 516536) was prepared by adding 50 mL 50 ml, Horse Serum ( GIBCO, 16050-122)), CO 2 incubator (5% CO ₂ , 37 ° C). The medium was changed every other day. A 0.25% trypsin-EDTA solution was used for cell detachment.

Nerve cells (PC-12) were inoculated in a DMEM medium to a 96-well plate (collagen coat) at 2 × 10 ³ cells / 100 μL / well and cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C.) , And the next day was replaced with medium containing the test substance (1% FBS, 10 ng / mL NGF (Alomone Labs, PRODUCT # N-100)). After incubation for 24 hours, amyloid beta (1-40) (SIGMA, Cat. No. A1075-.1MG) (PBS (Nissui Seiyaku Co., Ltd., Code 05913) (Concentration: 1 mM) and incubated at 37 占 폚 for 3 days to prepare a dissolution solution) was further added (50 占 퐇 / well) and cultured for another 24 hours. After the culture supernatant was removed from each well after completion of culture, 100 μL (per well) of test medium containing 10% live cell count measuring reagent (WST-8) (Nacalai Tesque, Cat. No. 07553-44) 30 min and 90 min. Subsequently, the absorbance (absorption wavelength 450 nm, reference wavelength 630 nm) of each well was measured with a microplate reader (Precision microplate reader (Molecular Devices)), the amount of absorbance change per hour was calculated from the positive values, , And viability of the cells was measured. In this example, selenonane-containing fish extracts: 2 mg / mL and selenonine: 100 nM were used as test substances (species concentrations).

The addition of amyloid beta resulted in about 70% viable cell count in the non-additive. When each test substance was treated under these conditions, the survival rate of cells was significantly improved in the selenolane-containing fish extract as compared with the control of addition of amyloid-β (FIG. 11).

Example 7: Review of dry eye improving effect

HCE-T cells (human immortalized corneal epithelial cells) (Ricken Bio Resource Center, RCB2280) were diluted in test medium (DMEM / F12 (ThermoFisher, Cat. No. 11330-032) , 1% NEAA (Gibco, Cat. No. 11140-050), 1% antibiotic (Nacalai Tesque, Cat. No. 26253-84) The cells were pre-cultured (pre-cultured) in a flask in a CO ₂ incubator (5% CO ₂ , 37 ° C) until the required number of cells reached. Cells were detached from the flask using a trypsin / EDTA solution (Nacalai Tesque, Cat. No. 32777-44), neutralized with trypsin, and centrifuged to recover the cells. Thereafter, the cells were resuspended in the test medium again and used as a cell suspension.

The selenenoine-containing fish extract was water-soluble and was directly dissolved in a medium to prepare a stock solution.

(1) Dry stress (dry eye) avoidance test in corneal epithelial cells

HCE-T was inoculated into a 96-well plate at 2.5 × 10 ⁴ cells / 0.1 mL / well and cultured for 5 hours in a CO ₂ incubator (5% CO ₂ , 37 ° C.) And replaced with a test medium containing the test substance. Thereafter, the cells were cultured for one day. Then, the culture supernatant was removed, and the cells were exposed in a dry state (dry stress treatment time: 10 minutes). Then, a special medium was added, and further cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C) for 3 hours. Cell proliferation was measured using a living cell count measuring reagent SF (Nacalai Tesque, Cat. No. 07553-44) (WST-8).

(2) Mucin gene expression analysis test

The HCE-T 6 × 10 ⁴ cells for a day at the sowing and /0.5 mL / well of 48-well plates, CO ₂ incubator in _{(5% CO 2, 37 ℃} ) after incubation, was replaced with a test medium comprising a matter piheom . Five hours after the addition of the test substance, total RNA was recovered from the cells, and mucin gene expression analysis was performed by real-time PCR (Light Cycler 96 System (Roche)) after cDNA synthesis. The gene to be analyzed: membrane-bound mucin gene MUC1, internal standard gene: GAPDH.

The selenium-containing composition of the present invention protects the drying stress of the corneal cells and suppresses the dryness of the dry eye because the selenium-containing fish extract significantly increases the viable cell count and mucin gene expression after the dry stress by the addition of the selenonine- (Figs. 12 and 13).

Example 8: Inflammation inhibitory effect

A variety of inflammatory responses involve a transcription factor called "NFκB." When a cell is stimulated by a cytokine such as TNF-α, the inactive NFκB is activated and an inflammatory response is induced. The expression of NFκ-B has been reported to be exaggerated in many diseases including allergic diseases and cancer (Chemistry and Biology, Vol. 47, No. 9, P. 602-604 (2009)). The test substance confirmed the anti-inflammatory effect by inhibiting the activity of NFκ-B induced by TNF-α. In the NFκ-B activity measurement system, a reporter gene was introduced into a target cell, and NFκB transcription activity by TNFα stimulation was measured by luciferase activity. The concentration of the test substance was determined to be selenonine (coarse tablets): 0.4 mg / mL (HEK293 cells) and selenonine (tablets): 100 nM.

HEK293 cells (RICEN BIO RESOURCE CENTER, RBC 1637, Lot. 20) were cultured in a basal medium (MEM, 10% FBS, 1% NEAA, MEM NEAA: Gibco, Cat. No. 11140-050, FBS: Biowest ., Cat No. S1820, Lot No. 516536, antibiotic:. Nacalai Tesque, Cat No. 26253-84 ) to use, and can require cells in the CO ₂ incubator _{(5% CO 2, 37 ℃} ) in T75 flasks The cells were detached from the flask using trypsin / EDTA solution (Nacalai Tesque, Cat. No. 32777-44), neutralized with trypsin, and then centrifuged The cells were then resuspended in the test medium and used as a cell suspension.

HEK293 cells 1.5 × 10 ⁴ cells were cultured in 1 days /0.1 mL / well of seeding a 96-well plate, and CO ₂ incubator in _{(5% CO 2, 37 ℃} ). The DNA vector (pGL4.32 [luc2P / NF-kB-RE / Hygro] vector (Promega, Cat. No. E8491)) was then added to the cells using a transfection reagent (Roche, Cat. No. 06366244001) (0.1 mL / well) supplemented with the test substance (concentration of fish extract containing selenonin: 0.4 mg / mL or selenonin species concentration: 100 nM) the following day, % CO ₂ for 2 hours. (5 μL per well) of 200 ng / mL TNF-α medium (10 μL / mL TNF-α (SIGMA, Cat. No. T0157-10UG) stock was added to the test medium) Concentration: 10 ng / mL. Thereafter, the cells were cultured at 37 ° C and 5% CO ₂ for 5 hours, and NF-κB was activated by TNF-α stimulation to promote induction of the luciferase gene. After 5 hours of culture, 30 μL of the culture supernatant was extracted from each well. 70 μL of luciferase assay reagent (Promega, Cat. No. E2610) was added to each well (total of 150 μL of culture supernatant), gently mixed and allowed to stand for 2 minutes. Thereafter, Relative Light Units (RLU: Relative Light Units) of each well were measured with a plate reader for luminescence measurement (luminometer: Thermoscan Flash, Varioscan Flash, model number 5250040). 200 mM ammonium pyrrolidine dithiocarbamate (APDC) (SIGMA, Cat. No. P8765-1G) was used as a positive control.

Both selenonine and selenonane-containing fish extract significantly inhibited NFκ-B activation by TNF-α addition. Allergies, arthritis, rheumatism, cancer, and chronic inflammatory conditions (Fig. 14).

From the foregoing, it will be appreciated that the specific embodiments of the disclosure are described herein for illustrative purposes, but it should be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, the present disclosure is not limited to the case of the appended claims. In addition, the claims have rights to the full scope and equivalents thereof.

Example 9: Examination of the effect of improving the glucose tolerance abnormality

Conventionally, the expression level of selenoprotein P (SELENOP) gene in human is correlated with insulin resistance. Treatment of hepatocyte and muscle cells with purified SELENOP induces abnormality of the insulin signal, (Misu H., et al., Cell Metabolism, 12, 483-495 (2010)), on the contrary, by knockout of the SELENOP gene and knockdown by RNAi to improve insulin sensitivity and glucose tolerance of the whole body in mice. In addition, 6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 3 (PFKFB3) is a positive regulator of the relevant glycolytic pathway and insulin signal transduction system, and PFKFB3 as an inhibitor In contrast, it was reported that activation of the Akt signal transduction system was observed in cells obtained by overexpression of the PFKFB3 gene, that glucose uptake stimulated by insulin, transposition of glucose transporter (GLUT4) to cell membrane and Akt signal transduction system were inhibited (Trefely S., et al., J. Biol. Chem., 290, 25834-25846 (2015)).

In this example, the effect of selenonine-containing fish extract on the expression amounts of SELENOP gene and PFKFB3 gene was examined. (DMEM (Nacalai tesque, Cat. No. 08456-65), 10% FBS (Biowest, Cat. No. S1820, Lot No. 516536), 1% using Penicillin-Streptomycin Mixed Solution (Nacalai tesque , Cat. No. 26253-84)] increase in the CO ₂ incubator _{(5% CO 2, 37 ℃} ) to use cells, 8 × a Petri dish having a diameter of 8 cm Cells were seeded at 10 ⁵ cells / 15 mL and then cultured. The next day (80% Confluent), the medium was exchanged with 15 mL medium containing (1 mg / mL) or without containing selenenoine-containing fish extract and cultured for 24 hours. After completion of the culturing, the cells were detached from the wells using 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, and the cells were washed with 1 x PBS (Code 05913, Nissui Seiyaku Co., Ltd., Code 05913) to remove the PBS, and then 50 μL of 2.5 g / l-Trypsin / 1 mmol / l-EDTA solution was added thereto, followed by reaction at 37 占 폚. Then, 450 μL of 10% FBS-containing PBS was added to each well to stop the trypsin reaction. The cell detachment solution was recovered, centrifuged at 500 x g for 10 minutes at room temperature to remove the supernatant, and the cells (precipitate) were recovered. Total RNA was recovered from the cells using an RNeasy Mico Kit (QIAGEN, Cat. No. 74004), and the RNA concentration and purification degree were measured by a spectrophotometer and a bioanalyzer (Agilent Technologies, Agilent 2100). Subsequent operations were performed using amplification products such as GeneChip WT Terminal Labeling and Controls Kit, amplification of total RNA samples, fluorescent labeling and quality check according to their instructions. Subsequently, the adjusted sample was hybridized to a DNA microarray (Affymetrix, GeneChip Human Genome U133 Plus 2.0 Array) using a GeneChip Scanner 3000 7G System (Affymetrix), washed and stained, and DNA after hybridization The microarray was scanned, the image file was converted into numerical data, and processing such as background subtraction, normalization and calculation of the expression ratio was performed.

The selenonane-containing fish extract suppressed the expression amount of the SELENOP gene to 1/3 or less and, on the contrary, increased the expression amount of the PFKFB3 gene more than twice as compared with the control without addition of the selenonine-containing fish extract, It was found that the composition had an improvement in abnormal glucose tolerance (Table 7).

Example 10: Examination of lipid metabolism abnormal improvement effect

27-Hydroxycholesterol (27HC) is a metabolite of cholesterol that is abundant in the blood, promotes the progression of atherosclerosis, and causes 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, 172-282 (2014)). In fact, in mice knocked out of the CYP7B1 gene, the 27HC concentration in blood and tissues increases 4-5 times compared to the wild type (Umetani M., supra). In this example, the effect on the expression amount of CYP7B1 gene of selenonane-containing fish extract was verified. Cells and samples were adjusted in the same manner as in Example 9, and data were collected and analyzed. The selenonane-containing fish extract showed an effect of improving the lipid metabolism abnormality in the selenium-containing composition of the present invention (Table 8) since the expression amount of the CYP7B1 gene was increased to 8 times or more as compared with no addition (control) .

The selenium-containing composition of the present invention can be used as an agent for reducing ischemia reperfusion injury, anti-metabolic syndrome, and the like. New uses of selenone concentrate extracted from fish processing residue are found and can contribute to high added value of low unused aquatic products. A new plant material or a functional material corresponding to a consumer's health orientation can be commercialized.

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

Claims (10)

Formula I:

Wherein R is hydrogen, an erthioyl group, a glutathionyl group, or a systeinyl group,
The present invention relates to a method for the prevention of metabolic syndrome including prevention of oxidative stress, prevention of oxidative lipid, prevention of ischemic diseases, improvement of metabolic syndrome, improvement of impaired glucose tolerance, prevention of metabolic syndrome including metabolic syndrome, A composition for inhibiting reduction of systemic immunity, inhibition of vascular endothelial dysfunction, inhibition of arteriosclerosis, prevention of dementia, improvement of brain function, protection of corneal epithelial cells, improvement of dry eye, or inhibition of inflammation. The process according to claim 1, wherein the organic selenium compound has the formula II:

3- (2-hydroxteleno-1H-imidazol-5-yl) -2- (trimethylammonio) propanoate. Formula III:

As an active ingredient, an effective amount of an organic selenium compound represented by the following formula (1): (1) an oxidative stress reduction, a lipid oxidation inhibition, an ischemic disease prevention, a metabolic syndrome improvement, an impaired glucose tolerance, A composition for inhibiting reduction of systemic immunity, inhibition of vascular endothelial dysfunction, inhibition of arteriosclerosis, prevention of dementia, improvement of brain function, protection of corneal epithelial cells, improvement of dry eye, or inhibition of inflammation. Formula IV:

The present invention relates to a method for the prevention of metabolic syndrome including prevention of oxidative stress, prevention of oxidative lipid, prevention of ischemic diseases, improvement of metabolic syndrome, improvement of impaired glucose tolerance, prevention of metabolic syndrome including metabolic syndrome, A composition for inhibiting reduction of systemic immunity, inhibition of vascular endothelial dysfunction, inhibition of arteriosclerosis, prevention of dementia, improvement of brain function, protection of corneal epithelial cells, improvement of dry eye, or inhibition of inflammation. A pharmaceutical composition comprising the composition according to any one of claims 1 to 4 for the treatment of metabolic syndrome including oxidative stress reduction, lipid oxidation inhibition, prevention of ischemic diseases, improvement of metabolic syndrome, improvement of glucose tolerance abnormality, A food composition for improving the protection of disorders, carcinogenesis inhibition, inhibition of reduction of systemic immunity, inhibition of vascular endothelial dysfunction, inhibition of arteriosclerosis, prevention of dementia, improvement of brain function, protection of corneal epithelial cells, improvement of dry eye or inhibition of inflammation. A food comprising the composition according to any one of claims 1 to 4. Formula I:

Wherein R is hydrogen, an erthioyl group, a glutathionyl group, or a systeinyl group,
The present invention relates to a method for the prevention of metabolic syndrome including prevention of oxidative stress, prevention of oxidative lipid, prevention of ischemic diseases, improvement of metabolic syndrome, improvement of impaired glucose tolerance, prevention of metabolic syndrome including metabolic syndrome, A pharmaceutical composition for suppressing the reduction of systemic immunity, inhibiting vascular endothelial dysfunction, inhibiting arteriosclerosis, preventing dementia, improving brain function, protecting corneal epithelial cells, improving dry eye or suppressing inflammation. 8. The process according to claim 7, wherein the organic selenium compound has the formula II:

(2-hydroxycarbonyl-1H-imidazol-5-yl) -2- (trimethylammonio) propanoate. Formula III:

As an active ingredient, an effective amount of an organic selenium compound represented by the following formula (1): (1) an oxidative stress reduction, a lipid oxidation inhibition, an ischemic disease prevention, a metabolic syndrome improvement, an impaired glucose tolerance, A pharmaceutical composition for suppressing the reduction of systemic immunity, inhibiting vascular endothelial dysfunction, inhibiting arteriosclerosis, preventing dementia, improving brain function, protecting corneal epithelial cells, improving dry eye or suppressing inflammation. Formula IV:

The present invention relates to a method for the prevention of metabolic syndrome including prevention of oxidative stress, prevention of oxidative lipid, prevention of ischemic diseases, improvement of metabolic syndrome, improvement of impaired glucose tolerance, prevention of metabolic syndrome including metabolic syndrome, A pharmaceutical composition for suppressing the reduction of systemic immunity, inhibiting vascular endothelial dysfunction, inhibiting arteriosclerosis, preventing dementia, improving brain function, protecting corneal epithelial cells, improving dry eye or suppressing inflammation.

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