JP7598656B2 - Selenium compound-containing composition - Google Patents

Description

The present invention relates to a composition containing a selenium compound as an active ingredient.

Selenium is an essential trace element that cannot be synthesized in the body, and is found in certain foods such as seafood, algae, meat, and egg yolks. Selenium plays an important role as an enzyme and antioxidant. For this reason, it has attracted attention as it is said to be linked to the prevention of aging. Selenoneine, described in Patent Document 1, is known as a compound containing selenium (hereinafter referred to as "selenium compound"). Selenoneine has strong antioxidant properties and exhibits biological antioxidant effects in humans.

Patent Document 1 discloses a composition containing selenoneine as an active ingredient for inhibiting the proliferation of colon cancer cells, inhibiting the formation of colon cancer, preventing non-alcoholic hepatitis, preventing Alzheimer's dementia, inhibiting inflammation accompanied by NFκ-B activation, protecting corneal epithelial cells by activating the membrane-bound mucin gene MUC1, inhibiting the expression of the selenoprotein P gene, improving impaired glucose tolerance, and inducing the expression of the CYP7B1 gene.

International Publication No. 2018/066676

Although Patent Document 1 describes various uses of selenium compounds, it is difficult to say that the usefulness of selenium compounds has been fully elucidated.

The present invention was made in consideration of these circumstances, and aims to provide a new use for a composition containing a selenium compound as an active ingredient.

As a result of conducting cell tests and clinical trials on selenium compounds, the inventors of the present application found that selenium compounds (i) have the function of inhibiting the oxidation of LDL (low density lipoprotein), (ii) have the function of inhibiting the adsorption of oxidized LDL to vascular endothelial cells, (iii) have the function of inhibiting the adsorption of oxidized LDL to macrophages, (iv) have the function of inhibiting the expression of LOX-1 (lectin-like oxidized LDL receptor), and (v) have the function of lowering VLDL (very low density lipoprotein) levels.

Here, atherosclerosis is initiated when vascular endothelium is damaged by various stresses, and LDL in the blood infiltrates the intima of the vascular endothelium. LDL is converted to oxidized LDL by the action of vascular endothelial cells, and oxidized LDL is adsorbed (received) by macrophages in the intima via LOX-1, promoting the expression of LOX-1. As a result, the adsorption of oxidized LDL to macrophages is accelerated, and the macrophages are transformed into foam cells. When these cells aggregate in the intima, plaques are formed, which aggravates arteriosclerosis. Therefore, according to the above-mentioned functions (i) to (iv), it is believed that selenium compounds can be used to inhibit the foam cell transformation of macrophages, which occurs due to the vicious cycle of LDL conversion to oxidized LDL, LOX-1 reception, LOX-1 expression induction, and promotion of LOX-1 reception, and further, it is believed that selenium compounds can be used to prevent or treat atherosclerosis.

Based on these findings, the present invention is a composition that contains a selenium compound as an active ingredient and is used to inhibit the adsorption of oxidized LDL to vascular endothelial cells, inhibit the adsorption of oxidized LDL to macrophages, inhibit LOX-1 expression, reduce VLDL levels, inhibit the transformation of macrophages into foam cells, or prevent or treat atherosclerosis.

The second invention is the first invention, in which the selenium compound is contained as a composition derived from fish meat.

The third invention is the first invention, in which the selenium compound is selenoneine.

The fourth invention is a food composition according to any one of the first to third inventions.

The fifth invention is a pharmaceutical composition according to any one of the first to third inventions.

Since the composition according to the present invention contains a selenium compound as an active ingredient, the composition can be used to inhibit the oxidation of LDL, inhibit the adsorption of oxidized LDL to vascular endothelial cells, inhibit the adsorption of oxidized LDL to macrophages, inhibit LOX-1 expression, reduce VLDL levels, inhibit the transformation of macrophages into foam cells, or prevent or treat atherosclerosis.

FIG. 1 is a chart showing the results of the antioxidant effect verification test. FIG. 2 is a graph showing the results of an oxidized LDL adsorption inhibition test using vascular endothelial cells (HUVEC). FIG. 3 is a chart showing the results of an oxidized LDL adsorption inhibition test using human monocyte cells (THP-1). FIG. 4 is a chart showing the results of a test for inhibiting oxidized LDL-induced LOX-1 expression. FIG. 5 is a graph showing the transition of neutral fat levels in the clinical trial as the amount of change from week 0. FIG. 6 is a graph showing the time course of RLP-C in the clinical trial, expressed as the amount of change from week 0. FIG. 7 is a graph showing the time course of VLDL, a precise measurement of lipoprotein fractions, in a clinical trial, as the amount of change from week 0. FIG. 8 is a graph showing the time course of quantitative determination of VLDL lipoprotein fraction in a clinical trial, expressed as the amount of change from 0 weeks. FIG. 9 is a graph showing the time course of 8-OHdG concentration in the clinical trial, expressed as the amount of change from week 0. FIG. 10 is a graph showing the time course of fasting blood glucose levels in the clinical trial, expressed as the amount of change from week 0.

The following describes in detail an embodiment of the present invention. Note that the following embodiment is merely an example of the present invention and is not intended to limit the scope of the present invention, its applications, or its uses.

[About the composition]
The present embodiment is a composition (hereinafter, sometimes referred to as "the present composition") for inhibiting the oxidation of LDL, inhibiting the adsorption of oxidized LDL to vascular endothelial cells, inhibiting the adsorption of oxidized LDL to macrophages, inhibiting LOX-1 expression, reducing VLDL levels, inhibiting the transformation of macrophages into foam cells, or preventing or treating atherosclerosis, which contains a selenium compound (organic selenium compound) as an active ingredient. The composition can be used, for example, by orally ingesting it, in a method for inhibiting LDL oxidation, inhibiting adsorption of oxidized LDL to vascular endothelial cells, inhibiting adsorption of oxidized LDL to macrophages, inhibiting LOX-1 expression, decreasing VLDL levels, inhibiting foam cell formation of macrophages, or preventing or treating atherosclerosis in a person who requires such inhibition (e.g., a person at risk of atherosclerosis or a patient with atherosclerosis). The composition can also be used in a method for preventing or treating other diseases caused by an increase in oxidized LDL, adsorption of oxidized LDL to vascular endothelial cells, adsorption of oxidized LDL to macrophages, LOX-1 expression, an increase in VLDL levels, or foam cell formation of macrophages.

The selenium compounds contained in the composition include selenoneine monomer represented by chemical formula (2), its tautomer represented by chemical formula (3), selenoneine oxidized dimer represented by chemical formula (4), and chemically modified selenoneine represented by chemical formula (1).

The compound represented by chemical formula (1) is an organic selenium compound in which the substituent R is bound to hydrogen, ergothioneine, glutathione, cysteine, acetylcysteine, homocysteine, methylmercury, or a thiol compound presumed to be produced in the body, and also includes metals and polymeric materials to which the organic selenium compound is bound via a selenol group.

The selenium compound represented by chemical formula (2) is 3-(2-hydroseleno-1H-imidazol-5-yl)-2-(trimethylammonio)propanoate.

The selenium compound represented by chemical formula (3) is 3-(2-selenoxo-2,3-dihydro-1H-imidazol-4-yl)-2-(trimethylammonio)propanoate.

The selenium compound represented by chemical formula (4) includes a dimer in which a diselenide is formed via a selenol group, with the basic unit being a compound having a molecular structure in which a selenol group and a trimethylammonium group are bonded to the imidazole ring of the oxidized dimer (3,3'-(2,2'-diselanediylbis(1H-imidazole-5,2-diyl))bis(2-(trimethylammonio)propanoate)).

In this composition, selenoneine is contained as a composition derived from fish meat (such as dark meat) or fish blood. Fish whose meat or blood is used as an ingredient of this composition include tuna, swordfish, bonito, mackerel, yellowtail, horse mackerel, sardines, and sea bream. Selenoneine may also be derived from organisms other than fish (for example, marine mammals including toothed whales, and microorganisms).

When mackerel is selected as the fish, the composition is a selenium compound-containing composition containing amino acids and selenium compounds, and the amino acid composition is, for example, an amino acid composition derived from mackerel. As mackerel, the genus Scombrella, the genus Maccaronia, the genus Scombrella, etc. of the order Perciformes and the family Scombridae can be used, and as the genus Scombrella, the genus Chub mackerel, the pied mackerel, and the scombrella can be used.

When fish meat is used as the material for this composition, unprocessed raw fish or pieces of fish meat obtained by cutting or butchering raw fish, or pieces of fish meat obtained by crushing raw fish or pieces of fish meat, etc. can be used. The material for this composition may include the head, bones, and internal organs of fish, but from the viewpoint of being rich in selenium compounds, it is preferable to include the edible parts of fish meat and internal organs, and it is even more preferable to include internal organs.

This composition can be obtained, for example, by carrying out step 1 of inactivating a proteolytic enzyme contained in fish meat (a material of this composition) containing selenium compounds to obtain a fish meat-derived composition containing selenium compounds, and step 2 of adding a specific enzyme to the fish meat-derived composition obtained in step 1 to subject it to an enzymatic reaction, and then inactivating the added enzyme to obtain a selenium compound-containing composition containing the selenium compounds. The method for producing this composition will be described in detail later.

When the composition is made into a food composition (including beverages), in addition to a selenium compound such as selenoneine, it may contain other ingredients commonly used in food and beverages (nutritional ingredients such as vitamins and minerals, food ingredients, or food additives, etc.). Other ingredients that can be used include ethanol or water as a solvent, sweeteners, flavorings, seasonings, coloring agents, preservatives, bulking agents, thickening agents, thickening stabilizers, antioxidants, bittering agents, acidulants, emulsifiers, strengthening agents, manufacturing agents, excipients, disintegrants, binders, lubricants, coating agents, and plasticizers.

The composition can be provided as a solid, semi-solid or liquid food, for example, as confectionery (cookies, jellies, etc.), bread, processed fish and meat products, processed livestock products, noodles, soups, sauces, side dishes, beverages (milk beverages, lactic acid bacteria beverages, soft drinks, vegetable beverages, powdered beverages, sports beverages, nutritional beverages, etc.). The composition can also be provided as health foods, functional foods, nutritional supplements, supplements, foods for specified health uses, foods for the sick, combination foods for the sick, or foods for the elderly. The composition can also be provided as medicines or quasi-drugs.

The composition can be in the form of a solid, liquid, powder, granules, paste, mousse, gel, jelly, tablet, or other form (dosage form). The composition can also be in the form of a packaged bag, container, capsule, or the like.

In this composition, for example, the selenium compound content is 1 μg Se/g or more, and the total selenium content is 5 μg/g or more. This composition can be used so that the daily intake for an adult is 1 to 10 μg Se of selenium compounds and 5 to 100 μg of total selenium. In this case, the number of times per day to achieve the daily intake may be one or multiple times.

[Regarding the manufacturing method of the present composition]
The method for producing the present composition (hereinafter, sometimes referred to as "the present production method") will be described.

As described above, this production method involves step 1 in which a fish meat-derived composition containing selenium compounds is obtained by inactivating a protein-degrading enzyme (protease) contained in fish meat, and step 2 in which a specific enzyme is added to the fish meat-derived composition obtained in step 1 to subject it to an enzymatic reaction, and then the added enzyme is inactivated to obtain a selenium compound-containing composition containing selenium compounds. By carrying out step 1, it is possible to efficiently extract selenium compounds in step 2 while suppressing unnecessary protein decomposition reactions and spoilage.

Specifically, in step 1, before the above-mentioned protease is inactivated, water (tap water, well water, distilled water, ion-exchanged water, etc.) is added to the fish meat as a raw material as necessary, and the fish meat is pulverized in a mixer to prepare fish meat fragments or a mixed slurry of fish meat fragments and water. The mixed slurry is then subjected to a heat treatment to inactivate the above-mentioned protease. After the heat treatment, a mixture or slurry of the solids and water constituting the fish meat is obtained. The liquid phase portion obtained by solid-liquid separation of the mixture or slurry can be used as the fish meat-derived composition to be used in step 2. In addition to heating, pressurization or pH change can also be used as a method for inactivating the above-mentioned protease.

In step 2, both a protease (enzyme 1) derived from bacteria such as Bacillus subtilis and a protease (enzyme 2) derived from microorganisms such as Aspergillus oryzae can be used as the above-mentioned specific enzyme. In step 2, enzymes 1 and 2 are added in sequence to the fish meat-derived composition obtained in step 1, and the fish meat-derived composition is subjected to an enzyme reaction. The order in which enzymes 1 and 2 are added may be enzyme 1, enzyme 2, or enzyme 2, enzyme 1, but the former order allows for more efficient extraction of selenium compounds from the fish meat-derived composition. After the fish meat-derived composition is subjected to the enzyme reaction, enzymes 1 and 2 are inactivated by heating, applying pressure, changing the pH, or other methods.

After step 2 is completed, a mixture or slurry of the solids and water constituting the selenium compound-containing composition is obtained. The liquid phase portion obtained by solid-liquid separation of the mixture or slurry may be used as the present composition, or the liquid phase portion may be concentrated and used as the present composition, or the liquid phase portion (or a concentrate of the liquid phase portion) may be dried (e.g., spray-dried) to form a powder and used as the present composition. In this embodiment, a composition with a high content of selenium compounds is obtained by carrying out an enzyme reaction in two stages in step 2 using enzymes 1 and 2.

Below, examples of this composition are described. Note that the present invention is not limited to these examples as long as it does not deviate from the gist of the invention. In addition, unless otherwise specified, the units and measurement methods described in these examples are in accordance with JIS standards.

In each test (cell test, clinical trial) in this example, "Saba Peptide (product name)" manufactured and sold by the applicant was used as the composition (test substance, test food). The specifications for this composition are total selenium 5.0 μg/g or more (test method: inductively coupled plasma mass spectrometry), selenoneine 1.0 μgSe/g or more (test method: liquid chromatography inductively coupled plasma mass spectrometry), moisture 8.0 wt% or less (test method: Karl Fischer method), protein 85.0-95.0 wt% (test method: semi-micro Kjeldahl method), lipid 0.5 wt% or less (test method: Soxhlet extraction method), and ash 6-12 wt% (test method: direct ash method).

[1. Antioxidant effect verification test]
In order to verify the antioxidant effect of the test substance, an antioxidant effect verification test was carried out. The test substance was a stock solution of the composition adjusted to 100 mg/mL using a growth medium as a solvent and sterilized by filtration.

Vascular endothelial cells (HUVEC (CELL APPLICATIONS, INC., product number CA20005n)) were seeded in a 24-well plate at 0.2 x ¹⁰⁵ cells/well. Then, the cells were cultured in a _CO2 incubator. After confirming that the cells were confluent, the culture supernatant was removed from the well, and 46 μL of LDL (2.175 mg/ml), 2.5 μL of 1 mM copper sulfate ( _CuSO4 ), 2.5 μL of DMSO (dimethyl sulfoxide), and 2.5 μL of the test substance were added to 450 μL of medium (final LDL concentration 0.2 mg/mL, final copper ion concentration 5 μM). Then, the cells were cultured in a _CO2 incubator, and the culture supernatant was collected after 0 hours, 2 hours, 4 hours, and 8 hours, and the concentration of oxidized LDL was measured. The concentration was measured using the Oxi Select Human Oxidized LDL ELISA kit (MDA-LDL Quantitation (malondialdehyde oxidized low-density lipoprotein (MDA-oxLDL) detection reagent) manufactured by Cell Biolabs, Inc.

This test was conducted in two cases with different concentrations of the test substance added (0.8 mg/mL, 0.16 mg/mL), and in two cases for the control group to which no test substance was added (no copper sulfate added, copper sulfate added). Figure 1 shows the measurement results of oxidized LDL concentration for each case.

According to the measurement results shown in Figure 1, the oxidation of LDL was significantly observed in the presence of copper sulfate, and when the test substance (the composition) was added, the oxidation of LDL was suppressed. The higher the concentration of the test substance, the greater the inhibition of the oxidation of LDL.

[2. Oxidized LDL adsorption inhibition test]
In order to verify the effect of the test substance on inhibiting the adsorption of oxidized LDL in vascular endothelial cells and macrophages, an oxidized LDL adsorption inhibition test was performed. The test substance was a stock solution of the composition, which was added to a cell culture medium as a solvent and stirred to a concentration of 100 mg/mL, and then sterilized by filtration. In addition to the control group, this test was performed on three cases with different concentrations of the test substance (0.1 mg/mL, 1 mg/mL, 10 mg/mL) and two cases with different types of positive target substances (0.5 mM Metformin hydrochloride (Abcam, product number ab146725) and 1 μM Chelerythrine chloride (Funakoshi, product number AG-CR1-0071-M001)).

<Tests on Vascular Endothelial Cells>
Vascular endothelial cells (HUVEC (PromoCell, product number D10013)) were seeded on a gelatin-coated 96-well plate at 1 x 10 ⁴ cells/well. After 24 hours of culture, the medium was replaced with a medium containing a test substance or a positive control substance, and cultured for another 24 hours. After that, the cells were washed with serum-free medium, and then replaced with a staining solution (a solution of fluorescently labeled oxidized LDL) containing 20 μg/mL Dil-OxLDL (Thermo Fisher, product number L34358) and 5 μg/mL nuclear staining dye (Hoechst33342 (DOJINDO, product number 346-07951)), and reacted for 30 minutes. After that, the cells were washed with PBS, and the fluorescence intensity and cell number of the cells were measured and quantified using a fluorescent plate reader (Thermo Fisher, Varioskan LUX) for quantitative analysis.

To quantify the fluorescence intensity, the fluorescence intensity (excitation wavelength 554 nm/fluorescence wavelength 571 nm) of each cell was measured using a fluorescence plate reader, and the number of cells was measured. Then, Dil-OxLDL/Hoechst was calculated as the adsorbed Dil-OxLDL value per cell, and the relative fluorescence intensity value was calculated from the value of the control group (untreated with Dil-OxLDL). Figure 2 shows the measurement results (relative fluorescence intensity values) for the test substance, positive control substance, and control group. On the horizontal axis of Figure 2, "0.1" represents the case of 0.1 mg/mL of the test substance, "1" represents the case of 1 mg/mL of the test substance, and "10" represents the case of 10 mg/mL of the test substance. This is the same in Figures 3 and 4.

According to the measurement results shown in Figure 2, the relative fluorescence intensity values were lower for all test substances than for the control group, confirming the effect of the test substances in inhibiting the adsorption of oxidized LDL to cells. For this composition, a significant difference was confirmed compared to the control group at 1 mg/mL and 10 mg/mL (p<0.05). It was confirmed that this composition has the effect of inhibiting the adsorption of oxidized LDL to cells.

<Macrophage Tests>
Human monocyte cells (THP-1 (RIKEN BioResource Center, product number RCB1189)) were seeded onto a 96-well plate at 1 x 10 ⁴ cells/well, and 0.5 μg/mL PMA (phorbol 12-myristate 13-acetate) was added at the same time as seeding, and the cells were cultured for 4 days in a CO ₂ incubator (5% CO ₂ , 37°C). After confirming that the cells had differentiated into macrophage cells (adherent cells), the medium was replaced with one containing the test substance or positive control substance, and the cells were cultured for 24 hours. Thereafter, the cells were washed with serum-free medium, and then replaced with a staining solution (fluorescently labeled oxidized LDL solution) containing 20 μg/mL Dil-OxLDL (Thermo Fisher, product number L34358) and 5 μg/mL nuclear staining dye (Hoechst33342 (DOJINDO, product number 346-07951)), and allowed to react for 30 minutes. Thereafter, the cells were washed with PBS, and the fluorescence intensity and cell number of the cells were measured and quantified using a fluorescence plate reader (Thermo Fisher, Varioskan LUX) for quantitative analysis. The method of quantifying the fluorescence intensity was the same as in the test for vascular endothelial cells. FIG. 3 shows the measurement results (relative fluorescence intensity values) for the test substance, positive control substance, and control group.

According to the measurement results shown in Figure 3, the relative fluorescence intensity values were lower for all test substances than for the control group, confirming the effect of the test substances in inhibiting the adsorption of oxidized LDL to macrophages. For this composition, a significant difference was confirmed compared to the control group at 10 mg/mL (p<0.05). It was confirmed that this composition has the effect of inhibiting the adsorption of oxidized LDL to macrophages.

[3. LOX-1 expression inhibition test]
To verify the inhibition of LOX-1 expression, a LOX-1 expression inhibition test was conducted. The same test substance and positive target substance as those in the oxidized LDL adsorption inhibition test were used in this test. In addition to the control group, this test was conducted on three cases with different concentrations of the test substance (0.1 mg/mL, 1 mg/mL, 10 mg/mL) and two cases with different types of positive target substances (0.5 mM Metformin hydrochloride (Abcam, product number ab146725) and 1 μM Chelerythrine chloride (Funakoshi, product number AG-CR1-0071-M001)).

Specifically, human monocyte cells (THP-1 (RIKEN Bioresource Center, product number RCB1189)) were seeded in a 96-well plate at 1 x 10 ⁴ cells/well, and 0.5 μg/mL PMA (Phorbol 12-myristate 13-acetate) was added at the same time as seeding, and the cells were cultured for 4 days in a CO ₂ incubator (5% CO ₂ , 37°C). After confirming that the cells had differentiated into macrophage cells (adherent cells), the medium was replaced with one containing the test substance or positive target substance, and the cells were cultured for 24 hours. Thereafter, the medium was replaced with one containing 50 μg/mL OxLDL (Thermo Fisher, product number L34358), and the cells were reacted for 24 hours, after which total RNA was collected from the cells. The total RNA was converted into cDNA, and the LOX-1 gene expression level was measured by real-time PCR (using Illumina's Realtime PCR ECO). FIG. 4 shows the results of measuring the expression level of the LOX-1 gene.

According to the measurement results shown in Figure 4, LOX-1 expression was lower in all test substances than in the control group, confirming the inhibitory effect of the test substances on LOX-1 gene expression. For this composition, a significant difference was confirmed compared to the control group at 10 mg/mL (p<0.05). This confirmed that this composition has the effect of inhibiting LOX-1 expression.

4. Clinical Trials
A clinical trial was conducted to examine the cholesterol-lowering effect and effect on arteriosclerosis of a food containing a selenium compound (the present composition).

- Testing method -
The clinical trial method used was a randomized, parallel-group, double-blind comparative study using two test food intake groups: a group consuming a selenium compound-containing food (test food group) and a group consuming a placebo food (control food group).

-subject-
Regarding the subjects of the study, the "Points to Note When Preparing an Application for Food for Specified Health Uses" for FOSHU states that it is desirable to target subjects with borderline LDL cholesterol levels, which are set as the target subjects for "cholesterol-related" studies, and that minors and pregnant women must be excluded. Therefore, taking these factors into consideration, the following eligibility and exclusion criteria were established. Eligibility criteria (1) to (6) were established with the objectives of the clinical trial in mind. Eligibility criteria (7) to (11) and exclusion criteria (1) to (4) were established due to ethical considerations regarding the subjects.

<Eligibility Criteria>
(1) Borderline and mild LDL cholesterol levels: LDL cholesterol 120-139 mg/dL
(2) Age: 30 to under 70 years old (3) Gender: No restrictions (however, to avoid bias in the male-female ratio, we will set the number of males and females in each group as equal as possible).
(4) Non-smokers. (5) Non-users of foods high in selenium compounds (fish such as mackerel). (6) Non-users of supplements or health foods (including those containing selenium compounds). (7) Non-suffering from lifestyle-related diseases (high blood pressure, diabetes, etc.), rheumatism, liver disorders, kidney disorders, or other chronic diseases. (8) Non-treatment history of malignant tumors, heart failure, or myocardial infarction. (9) Non-allergic to fish high in selenium compounds, medicines, or foods high in red koji, or medicines. (10) Non-medicinal visits or medications for the purpose of treatment. (11) Non-persons who fully understand the contents of the clinical trial and have given their written consent.

<Exclusion Criteria>
(1) Pregnant or breastfeeding women, or those who intend to become pregnant during the study period. (2) Those who are currently participating in other clinical trials or clinical studies, or those who have participated in other clinical trials or clinical studies within the past three months. (3) Those who are unable to follow the instructions of the doctor in charge or medical institution staff. (4) Others who are deemed to have some kind of problem by the study administrator.

In this study, 31 subjects were evaluated for eligibility, 19 were excluded, and 12 subjects who met the above eligibility criteria and did not violate the exclusion criteria were included as subjects. The 12 subjects were then divided into 6 subjects in the test food group and 6 subjects in the control food group. The subjects were randomly assigned using the permuted block method. There were no deviations from the protocol. Interviews with the subjects during the study period revealed no problems with their physical condition, no adverse events, and the study was conducted entirely according to the protocol. The food intake rate was over 80% in all cases, and no dropouts were observed during the study. No significant differences were observed between the test food group and the control food group in the subjects' background data for any of the following items: BMI, systolic blood pressure, diastolic blood pressure, pulse rate, or body temperature.

- Test food and method of taking the test food -
The test food was a food containing 1000 mg of the present composition (total selenium 5.0 μg/g or more, selenoneine 1.0 μgSe/g or more) (per 4 tablets) for the test food group, and 0 mg of the present composition (per 4 tablets) for the control food group. The test food was stored at room temperature, and a 12-week supply was given to each subject on the day of the first examination. Any remaining test food was collected from the subjects on the last day of the study period. The test food was blinded, and given to the subjects based on the identification number assigned to the food. All people involved in the study (subjects, interventionists, evaluators, etc.) were blinded, and the allocation list was not opened until the subjects to be analyzed were determined.

Each subject was instructed to (i) take the tablets with water immediately after breakfast or dinner (within 30 minutes) during the intake period, (ii) refrain from excessive eating and drinking during the intake period, although no specific dietary requirements were specified, and (iii) take the tablets immediately after dinner if they forgot to take the tablets immediately after breakfast, and take the tablets before going to bed if they forgot to take the tablets after dinner. However, because the nature of the test food makes it desirable to take the tablets with meals, subjects were instructed to take the tablets immediately after breakfast or dinner (within 30 minutes) as a general rule.

- Inspection Schedule -
Each subject visited the medical institution (study institution) according to the schedule shown in Table 1, and underwent the tests, interviews, and measurements listed in the test items. For this schedule, the date of visit at week 0 of intake was counted as the starting date, and tests, etc. were performed at 6 weeks of intake (6 weeks: within 42 days ± 6 days from the date of the first test) and 12 weeks of intake (12 weeks: within 84 days ± 6 days from the date of the first test). Note that if a patient visited the hospital within 42 days ± 6 days at 6 weeks of intake or within 84 days ± 6 days at 12 weeks of intake for an examination, it was considered that this would have little effect on the study results, and so it was treated as an example of protocol compliance.

In the blood test (general items), the hematological test items selected for measurement were white blood cell count, red blood cell count, hematocrit, MCV, MCH, MCHC, and platelet count, and the blood biochemistry test items selected for measurement were AST, ALT, γ-GTP, ALP, LDH, total bilirubin, albumin, total protein, blood glucose level, HbA1c, urea nitrogen, creatinine, GFR, uric acid, sodium, potassium, calcium, total cholesterol, HDL cholesterol, LDL cholesterol, and triglyceride (TG). In the blood test (general items) at screening, LDL cholesterol was measured as a screening test, and in the blood test (general items) at 12 weeks of intake, the concentration of selenoneine in the blood was measured.

For blood tests (items related to effectiveness), the following items were selected for measurement: total cholesterol, HDL cholesterol, LDL cholesterol, non-HDL cholesterol, triglycerides (TG), remnant-like lipoprotein cholesterol (RLP-C), apolipoprotein B, precise measurement of lipoprotein fractions (VLDL, IDL, LDL, HDL), and LOX-index. For measurement items that overlap with blood tests (general items), one measurement result was recorded for each test.

The following items were measured in the urine test: urinary protein, urinary glucose, urobilinogen, bilirubin, ketone bodies, specific gravity, pH, and 8-OHdG.

-Inspection details-
In order to accurately measure cholesterol levels, the subjects refrained from excessive eating and drinking the day before, and on the day of the test, they woke up and had breakfast (to ensure that the subjects were not hungry), and then underwent a medical interview, physical examinations, blood pressure measurements, and blood tests.

In the first test, the doctor in charge interviewed the subjects, confirmed that they met the inclusion criteria (including LDL cholesterol at screening), and then conducted blood and urine tests (first blood and urine collection). The subjects were given test foods with identification numbers A or B, and were instructed to take a total of four tablets of the test foods at breakfast or immediately after dinner starting the day after blood collection (it was acceptable to take the test foods once immediately after breakfast or dinner, or twice in separate doses, once immediately after breakfast and once immediately after dinner). The subjects were also given a daily diary (from week 0 to week 6) and instructed to record the intake of the test foods and the amount eaten every week. They were instructed to bring the completed diary with them on the day of the second test.

The second test was conducted 42 days after the first test (6 weeks after the first test). On the day of the test, the doctor in charge interviewed the participants and performed a blood test (second blood draw). The daily diary (from weeks 0 to 6) was collected, and a new diary (from weeks 7 to 12) was handed out. Participants were instructed to record their intake of the test foods and the amount eaten every week, and to bring it with them on the day of the third test.

The third test was conducted 42 days after the second test (12 weeks after the first test). On the day of the test, the doctor in charge interviewed the subjects and performed a blood test (third blood draw). The test foods were collected from the subjects, and daily diaries (from weeks 7 to 12) were also collected. It was confirmed that there were no problems with the consistency of the collected test foods with the daily diaries.

-Statistical analysis method-
The actual measured values, amount of change, and rate of change for each data were used as analysis data. Statistical methods used included Student's t-test for between-group comparisons. Paired t-tests were used for within-group comparisons with pre-ingestion values. The significance level was set at 5% on both sides. SPSS Statistics 28.0.1 (IBM Japan) was used as statistical analysis software.

- Analysis results -
5 to 10 show graphs of the test items for which positive results were obtained in the test food group (triglycerides (FIG. 5), RLP-C (FIG. 6), precise measurement of lipoprotein fraction VLDL (FIG. 7), lipoprotein fraction VLDL quantification (FIG. 8), 8-OHdG concentration (FIG. 9), and fasting blood glucose (FIG. 10)). In this test, a significant difference was observed at 6 weeks for very low density lipoprotein (VLDL), as shown in FIG. 7-8, compared to the control food group, and a significant tendency was observed at 6 weeks for RLP-C, as shown in FIG. 6. In addition, although no significant difference was observed for 8-OHdG, as shown in FIG. 9, a downward tendency was observed in the test food group throughout the 12 weeks. Although not shown in the figures, significant differences were also observed in LH ratio and apolipoprotein B at 6 weeks. In this test, it was confirmed that the composition has the effect of lowering the level (concentration) of VLDL.

The present invention can be applied to compositions that contain selenium compounds as active ingredients.

Claims (4)

A composition for inhibiting the adsorption of oxidized LDL to vascular endothelial cells, which comprises selenoneine as an active ingredient, even when the oxidation of LDL is not inhibited . The composition according to claim 1 , wherein the selenoneine is contained as a composition derived from fish meat or fish blood. The composition according to claim 1 or 2 , wherein the composition is a food composition. The composition of claim 1 or 2 , wherein the composition is a pharmaceutical composition.

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