TWI810239B - Food composition for improvement or prevention of lifestyle-related diseases - Google Patents

Abstract

The present invention provides a food composition for improving or preventing lifestyle-related diseases, characterized in that fulvic acid is an active ingredient; a food composition for improving liver function, characterized in that fulvic acid is an active ingredient; a food composition for anti-fatigue A substance characterized by fulvic acid as an active ingredient; a food composition for muscle strengthening characterized by fulvic acid as an active ingredient; and a mitochondrial activator characterized by fulvic acid as an active ingredient.

Description

Food composition for improvement or prevention of lifestyle-related diseases

The present invention relates to a food composition for improving or preventing lifestyle-related diseases, a food composition for improving liver function, a food composition for anti-fatigue, a food composition for muscle strengthening, and a mitochondrial activator.

In recent years, due to Westernized eating habits, lifestyle-related diseases such as increased fat intake, hyperlipidemia, hypertension, diabetes, and obesity have gradually increased. In general, factors for the occurrence and prognosis of lifestyle-related diseases include, for example, lifestyle habits, heredity, and environment. In addition, lifestyle habits include, for example, eating habits, exercise habits, rest, smoking, drinking, and the like.

Humic substances are mainly plant residues and microbial remains in the soil. The carbohydrates and proteins in them are decomposed by microorganisms over many years, and the decomposition products are condensed into polymers. Humic substances are amorphous polymers containing fulvic and humic acids. Humic acid is a reddish brown or dark brown amorphous high molecular organic acid that is soluble in alkali and insoluble in acid. On the other hand, fulvic acid is an amorphous polymer organic acid soluble in alkali and acid. Humic substances, in addition to fulvic acid and humic acid, are also known to contain fatty acids, organic acids, amino acids, proteins, minerals, etc.

Such fulvic acid (FA: Fulvic acid) is an organic acid derived from humus soil. According to the classical medical treatment of India: Ayurveda (Ayurveda), unrefined humus is used as "Shilajit" is used. Accumulation of leaf humus containing fulvic acid has been observed in some parts of the United States and Saga Prefecture in Japan, and the industry is increasingly moving towards the standardization of fulvic acid.

The health benefits of shilajit containing fulvic acid are derived from the high concentration of fulvic acid. It can be used as an oral medicine to prevent cognitive impairment, Alzheimer's disease, respiratory tract infection, cancer, heavy metal toxicity, eczema, and Medication for hypoxia. However, there is still no supporting evidence, and the mechanism of action of fulvic acid is completely unknown.

Also, regarding humic acid, for example, it has been reported in Non-Patent Document 1 that it is associated with an anti-inflammatory action.

[Prior technical literature] 【Non-patent literature】

[Non-Patent Document 1] Phytother Res. 2015 Jun; 29(6): 791-5

The object of the present invention is to provide a food composition which has the effects of improving or preventing lifestyle-related diseases, improving liver function, anti-fatigue, and strengthening muscles, and the object of the present invention is to provide a mitochondrial activator.

As a result of continuous in-depth research by the inventors in order to achieve the above-mentioned purpose, it was found that fulvic acid It can promote the uptake of carbohydrates in peripheral tissues, has the effect of muscle differentiation, protects the mitochondrial membrane potential and activates the ATP consumption system. Mitochondrial prestored oxygen consumption capacity, considered an important aspect of mitochondrial function, is defined as the difference between basal ATP production and its maximal activity. When cells are under stress, the ATP necessary to maintain cell function increases, and the energy demand increases. Cells with greater pre-stored oxygen-consuming capacity can generate more ATP and thus overcome more stress including oxidative stress. Furthermore, oral intake of fulvic acid in mice was shown to suppress weight gain due to a high-fat diet, which indicates that it has a significant effect on lifestyle-related diseases. In addition, with regard to the liver function of mice, it can be seen from the results of blood tests that the liver function can be improved by fulvic acid.

The present invention is completed based on these knowledge and further repeated examinations, and provides the following food compositions for improving or preventing lifestyle-related diseases, food compositions for improving liver function, food compositions for anti-fatigue, and food compositions for strengthening muscles , and mitochondrial activators.

Item 1. A food composition for improving or preventing lifestyle-related diseases, characterized in that fulvic acid is an active ingredient.

Item 2. The food composition as described in Item 1, wherein the lifestyle-related disease is selected from the group consisting of diabetes, insulin resistance, hyperlipidemia, obesity, fatty liver, dyskinesia, sarcopenia, and cachexia At least 1 type in the constitution group.

Item 3. A food composition for improving liver function, characterized in that fulvic acid is an active ingredient.

Item 4. An anti-fatigue food composition characterized in that fulvic acid is an active ingredient.

Item 5. A food composition for muscle strengthening, characterized in that fulvic acid is an active ingredient.

Item 6. A mitochondrial activator characterized in that fulvic acid is an active ingredient.

Item 7. A composition for activating mitochondria, characterized in that fulvic acid is an active ingredient.

Item 8. A method for improving or preventing lifestyle-related diseases, a method for improving liver function, a method for improving fatigue, a method for strengthening muscles, or a method for activating mitochondria, which is characterized by comprising administering an effective amount of fulvic acid to mammals step.

Item 9. A use characterized in that it is a food composition for improving or preventing lifestyle-related diseases, a food composition for improving liver function, a food composition for anti-fatigue, a food composition for muscle strengthening, or a mitochondrial activator In the manufacture of fulvic acid is used.

Item 10. A use characterized by use of fulvic acid as an agent for improving or preventing lifestyle-related diseases, an agent for improving liver function, an agent for anti-fatigue, an agent for strengthening muscles, or an agent for activating mitochondria.

Item 11. The method as described in Item 8, the use as described in Item 9, or the use as described in Item 10, wherein the aforementioned lifestyle-related disease is selected from diabetes, insulin resistance, hyperlipidemia, obesity, fat At least one of the groups of liver, dyskinesia, sarcopenia, and cachexia.

Fulvic acid has remarkable effects of improving or preventing lifestyle-related diseases, improving liver function, anti-fatigue, strengthening muscles, and activating mitochondria, so it can be effectively used as a food for improving or preventing lifestyle-related diseases Composition, food composition for improving liver function, food composition for anti-fatigue, food composition for muscle strengthening, and active ingredient of mitochondrial activator.

【Figure 1】Showing the effect of "Humicle" on promoting the uptake of carbohydrates by muscle cells chart. GLUT inhibitor 10μM (GLUT-I), 0.1% humic mineral, 1μM insulin. Data represent mean ± S.E.M. n=6

[ Fig. 2 ] Photographs showing the muscle differentiation-promoting effect of humic mineral obtained by immunohistochemistry.

[ Fig. 3 ] A graph showing the effect of humic minerals on muscle cell differentiation markers. Data represent mean ± S.E.M. n=6

[Fig. 4] A graph showing the time-dependent (top) and dosage response (bottom) of the ATP consumption-promoting effect of humic minerals. Data represent mean ± S.E.M. n=6

[FIG. 5] Photographs showing changes in mitochondrial membrane potential caused by humic minerals. (A) control group, (B) humic mineral 0.1%, (C) actinomycin 10μm

[ Fig. 6 ] A diagram showing the action of increasing mitochondrial membrane potential by humic minerals. (Left) Carrier, (Right) Humic Mine

[ Fig. 7 ] A graph showing the amount of exercise 30 to 60 minutes after administration of humic mineral to mice. ^* P<0.05, vs. vehicle, NS; not significant, n=6

[ Fig. 8 ] A graph showing the effect of reducing body weight by humic minerals when mice were fed a high-fat diet. Give ND: normal diet (CRF-1; Oriental Yeast Co., Ltd.), HFD: high fat diet (HFD-32; Japan Claire Co., Ltd.), HFD+humic mineral (with HFD -32 mixed feed). Data represent mean ± S.E.M. n=5

[ Fig. 9 ] A graph showing the effect of humic minerals on blood markers when mice were fed a high-fat diet. HFD (white tube column), HFD+humic ore (black tube column), the data represent mean±SEM. ^* P<0.05, ^** P<0.001, n=5

[Figure 10] shows the autopsy results (A, C) and pathological analysis of mice fed a high-fat diet Photographs of results (B, D) (fed for 6 weeks). (A, B) Liver follicles, (C, D) Epididymis, scale bar: 50 μm

[ Fig. 11 ] A graph showing the diameter (pixel) of adipocytes. Data represent mean ± S.E.M.

[ Fig. 12 ] A graph showing the results of a glucose tolerance test after mice were fed a high-fat diet for 6 weeks. Data represent mean ± S.E.M. n=5

[ Fig. 13 ] A graph showing the results of measurement of the ATP/ADP ratio of mouse muscle tissue (A) and the amounts of ATP and ADP per muscle homogenate protein (B). ^* P<0.05, vs. HFD group, n=4

[ Fig. 14 ] A graph showing the dose-response of the ATP consumption-promoting effect of fulvic acid. Data after 30 minutes of exposure. Data represent mean ± S.E.M. n=8

[ Fig. 15 ] A graph showing the analysis results of fulvic acid by Flux Analyzer. n=7

[ Fig. 16 ] A graph showing the analysis results of fulvic acid by Flux Analyzer. The left column represents the carrier, the central column represents fulvic acid, and the right column represents humic minerals. ^** P<0.001 vs carrier control, n=7

[ Fig. 17 ] A graph showing the infrared absorption spectra of humic minerals and standard substances.

[ Fig. 18 ] A diagram showing three-dimensional fluorescence spectra of humic minerals and standard substances.

Hereinafter, the present invention will be described in detail.

Also, "comprise" in this specification includes the meaning of "essentially consist of" and the meaning of "only consist of".

The food composition for improving or preventing lifestyle-related diseases, the food composition for improving liver function, the food composition for anti-fatigue, the food composition for muscle strengthening, and the mitochondrial activator of the present invention are characterized in that fulvic acid as an active ingredient.

Fulvic acid, which is contained in the substances contained in humic substances, is soluble in alkali and acid and has an amorphous high Molecular organic acids. The humic substance used as the raw material of fulvic acid is not particularly limited in terms of its origin and state, and any humic substance can be used.

Such humic substances include, for example, human shale (humic shale) obtained from ancient plant deposits (shale layers) in Emory County, Utah, USA. Humic shale is the fertile soil containing most nutrients from about 70 to 127 million years ago, which is not denatured into oil and coal, but remains as organic matter. The plants inhabiting the aforementioned era were buried underground during the Ice Age, transformed into sphagnum moss by microorganisms such as bacteria, and new organic matter was repeatedly accumulated on it, and decomposed by bacteria again, forming over time Stackers. The humic substances contained in the formed accumulation layer are mainly composed of organic polymers containing fulvic acid and humic acid, and more than 70 kinds of trace minerals.

The method for extracting fulvic acid from humic substances is not particularly limited, and various known methods can be used. Extraction means include, for example: solid-liquid extraction, liquid-liquid extraction, immersion, leaching, decoction, circulation extraction, supercritical fluid extraction, microwave extraction, mixing and stirring, and the like. The extraction means may be performed alone or in combination of two or more.

In the case of extracting fulvic acid by mixing humic substances with an extraction solvent, the extraction solvent can include, for example: water (for example, distilled water, ion-exchanged water, reverse osmosis water, etc.); alcohols (for example, methanol, ethanol, propanol etc.); mixtures thereof, etc. The extraction time, extraction temperature, pH of the extraction solvent, and the like in the extraction operation can be appropriately set in consideration of extraction efficiency and the like.

The extract of humic substances can be filtered in order to remove unnecessary substances and sterilize. The means of filtration is not particularly limited, and can be performed by conventional methods.

The extract of humic substances can be further processed for separation or purification of fulvic acid. Such separation or purification treatment includes, for example, concentration, concentration under reduced pressure, distillation, fractional distillation, Phase transfer, solvent extraction, crystallization, recrystallization, chromatography, etc. These methods may be performed alone or in combination of two or more.

Fulvic acid can be used in an unseparated or purified state (crude extract, etc.), or can be used in a separated and purified form.

In addition, fulvic acid can be used in free state or salt state. Salts of fulvic acid include, for example: inorganic salts of sodium, magnesium, potassium, calcium, aluminum, etc.; salts linked to organic bases such as methylamine, ethylamine, and ethanolamine; Basic amino acid-linked salts and ammonium salts of arginine and the like. The salt can be an acid salt, and related salts, specifically, include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; formic acid, acetic acid, propionic acid, oxalic acid, Organic acids such as malonic acid, malic acid, tartaric acid, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid, ethanesulfonic acid; acids with aspartic acid, glutamic acid, etc. Acid salt of amino acid linkage. Fulvic acid also includes those complexed with hydrates, solvates, and minerals.

The form of fulvic acid is not particularly limited, and examples thereof include powder, granule, solid, solution, suspension, emulsion, concentrated liquid, and slurry.

For fulvic acid, you can use your own preparations or commercially available products. Commercial products of fulvic acid include, for example, "Humicle (trademark) HC fulvic acid powder" sold by Style and Value Japan Co., Ltd. The fulvic acid contained in the humic ore has infrared absorption peaks at wavelengths of 3362, 2875, 1675, 1559, 1360, 1200, 1047 and 835 cm ^-1 in the infrared absorption spectrum of the FT-IR method (refer to Japanese Patent No. 6120342).

The mitochondrial activator of the present invention also includes the meaning of food composition.

Food composition includes any food and drink that mammals (including humans) can ingest, for example: dairy products; fermented foods (yogurt, cheese, etc.); beverages (coffee, fruit juice, Cocoa, tea drinks, sports drinks, refreshing drinks such as nutrition drinks, milk drinks, lactic acid bacteria drinks, drinks containing lactic acid bacteria, yogurt drinks, carbonated drinks, such as Japanese wine, foreign wine, fruit wine, etc.); Starch cream, etc.); Spreads (jams, etc.); Western-style desserts (chocolate, donuts, pies, cream puffs, chewing gum, fudge, jelly, candy, biscuits, cakes, puddings, biscuits, etc.) ; Ice desserts (ice cream, ice candy, ice cream, etc.); food (curry, beef bowl, miso soup, soup, meat sauce, pasta, pickles, dipping sauce, ham, sausage, bacon, etc.); Seasonings (salad dressing, pine, umami seasoning, soup cubes, miso, soy sauce, sauces, ketchup, oyster sauce, etc.), etc.

The preparation method of the food composition is also not particularly limited, and may be based on a suitable known method.

The food composition includes, for example, health food, functional food, nutritional supplement food, nutritional supplement, health food, food for specific health use, nutritional functional food, functionally indicated food, and the like. The dosage unit form when used as a nutritional supplement is not particularly limited and can be appropriately selected, and examples thereof include tablets, capsules, granules, liquids, powders, and the like.

In addition, the food composition also includes the meaning of an additive imparting an effect of improving or preventing lifestyle-related diseases, effect of improving liver function, effect of anti-fatigue, effect of strengthening muscles, and effect of activating mitochondria.

Food composition, if necessary, can be mixed with excipients, vitamins, minerals, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants Debonding agent, lubricant, dyeing agent, perfume, stabilizer, preservative, sustained release regulator, surfactant, gloss agent, solvent, wetting agent, etc.

The proportion of fulvic acid contained in the food composition, for example: 0.01~99% by mass %, 1~80 mass%, 10~70 mass%, etc.

The intake amount of the food composition can be appropriately set according to various conditions such as the body weight, age, sex, and symptoms of the ingester.

As shown in the test examples described below, the present inventors found that fulvic acid can promote the uptake of carbohydrates in peripheral tissues, has muscle differentiation effects, protects mitochondrial membrane potential, and activates the ATP consumption system. That is, the improvement or preventive effect against diabetes can be expected by promoting the uptake of carbohydrates in peripheral tissues. In addition, it has a muscle differentiation effect, and can be expected to improve or prevent muscle strengthening effects, sarcopenia, dyskinesias, and the like. Protecting the mitochondrial membrane potential and activating the ATP consumption system can be expected to improve mitochondrial aging and fatigue recovery.

Furthermore, it was found that by orally administering fulvic acid to mice, body weight gain caused by a high-fat diet can be suppressed, so an improvement or preventive effect against lifestyle-related diseases can be expected. In addition, in the glucose tolerance test of mice, it was observed that the intake of fulvic acid lowered the blood sugar level, and the effect on insulin resistance can be expected. Furthermore, regarding the blood test results of liver function and lipid metabolism in mice, it was observed that liver function and lipid metabolism were improved by fulvic acid. Furthermore, fulvic acid can activate appetite and increase the metabolism of muscle tissue (ATP/ADP ratio), and can be expected to improve or prevent cachexia constitution. In addition, since the function of mitochondria is enhanced, activation of mitochondria can be expected.

Therefore, fulvic acid has significant effects of improving or preventing lifestyle-related diseases, improving liver function, anti-fatigue, strengthening muscles, and activating mitochondria, so it can be suitable for improving or preventing lifestyle-related diseases. Food composition, food composition for improving liver function, food composition for anti-fatigue, food composition for muscle strengthening, and active ingredient of mitochondrial activator.

Here, the lifestyle-related disease is not particularly limited, and examples thereof include diabetes, insulin resistance, hyperlipidemia, hypertension, obesity, fatty liver, dyskinesia, sarcopenia, and cachexia ( dyscrasias), etc.

In this specification, muscle enhancement includes the meanings of increasing muscle mass, inhibiting (preventing) the decrease of muscle mass, and recovering the decreased muscle mass.

In addition, fulvic acid is highly safe because it is a natural ingredient.

【Example】

Hereinafter, examples are given in order to further describe the present invention in detail. However, the present invention is not limited to these Examples and the like.

animal

Unless otherwise specified, C57BL/6N male mice were used for the following experiments. 8-week-old C57BL/6N male and mouse lines were purchased from Shimizu Laboratory Materials Co., Ltd. (Kyoto, Japan).

Test Example 1: In Vitro Test (Promoting Effect of Humic Mineral on Carbohydrate Uptake in Muscle Cells)

According to conventional methods, C2C12 cells were differentiated. Replaced to glucose-free medium, after 30 minutes in the presence or absence of GLUT inhibitors, stimulated by 0.1% humic mineral or insulin. Glucose uptake was measured using Glucose Uptake-Glo (trademark) Assay (Promega, Madison, WI) to measure luminescent activity.

The result is shown in Figure 1. It can be seen from Figure 1 that although insulin can promote the intake of carbohydrates, 0.1% humic mineral can promote the intake of carbohydrates more strongly than insulin. In addition, part of its role is mediated by glucose transporter, which can be known by the preparation test of GLUT-I.

Test Example 2: In vitro test (muscle differentiation promoting effect of humic mineral)

According to conventional methods, C2C12 cells were differentiated. Stimulated by 0.1% humic mineral, and stained by MHC (Myosin heavy chain) antibody (mouse monoclonal MF20 antibody (Developmental Hybridoma Bank, Iowa City, IA)).

The result is shown in Figure 2. It can be seen from Figure 2 that the positive reaction of MHC antibody can be clearly induced by 0.1% humic mineral. That is, 0.1% humic mineral can promote muscle differentiation.

Mouse striated muscle-derived cells C2C12 were inoculated into a 6-well plate, and the final concentration of humic mineral was 0.1%, and cultured with differentiation medium for 5 days. Media exchange was performed every other day. Prepare total RNA according to conventional methods and perform qPCR analysis.

The result is shown in Figure 3. It can be seen from Figure 3 that 0.1% humic mineral can inhibit the discovery of early differentiation markers and induce late differentiation markers MCK (muscle creatine kinase).

Test Example 3: In Vitro Test (Promoting Effect of Humic Mineral on ATP Consumption)

Mouse striated muscle-derived cells C2C12 were inoculated into 96-well plates at a rate of 1.25×10 ⁴ cells/well, so that the final concentration of humic minerals was 0.1%, and the ATP content in the cells was measured over time. Cells were lysed at the times indicated in Figure 4, as determined using the ENLITEN (trademark) ATP Assay System (Promega, Madison, WI) with a 20/20n Luminometer (Turner BioSystems, Sunnyvale, CA). In addition, the concentration-dependent ATP consumption hyperactivity of humic minerals after 6 hours was also determined.

The result is shown in Figure 4. It can be seen from Figure 4 that the ATP consumption system can be driven by 0.1% humic ore. Although the data is not shown, the humic minerals at various concentrations did not show cytotoxicity.

Test Example 4: In Vitro Test (Membrane Potential Hyperactivity of Humic Ore Mitochondria)

C2C12 cells were inoculated into a 48-well plate at 1×10 ⁵ cells/well, and the final concentration of humic mineral was 0.1%. The mitochondrial membrane potential after 24 hours was observed with JC-1 pigment.

The result is shown in Figure 5. It can be seen from Figure 5 that 0.1% humic mineral did not affect the mitochondrial membrane potential after 24 hours, but the anticancer agent actinomycin did.

C2C12 cells were inoculated so that the final humic mineral concentration was 0.1%, and the mitochondrial membrane potential after 48 hours was measured using the Muse (trademark) Mitopotential Assay Kit (EMD Millipore Corp., Darmstadt, Germany).

The result is shown in Figure 6. It can be seen from Figure 6 that the mitochondrial membrane potential can be increased by 0.1% humic mineral.

Test Example 5: In vivo test (oral changes in behavior of mice caused by oral administration of humic minerals)

The mice in a cylinder with a diameter of 20 cm were scanned with a camera (Smart3.0 system, Bioresearch Center, Aichi, Japan). Oral administration of 200 μL of PBS (vehicle) and 100 mg/200 μL of humic minerals to ddY mice and ADX ddY mice from which the adrenal glands were removed was observed for 90 minutes. Compare the amount of spontaneous exercise in the 30-60th time period when the action is most different.

The result is shown in Figure 7. It can be seen from Figure 7 that the ATP consumption system can be driven by 0.1% humic mineral, and the possibility of activating the sympathetic nervous system is extremely high. When the sympathetic nervous system is activated, the peristaltic movement of the bowel is reduced, thereby inhibiting defecation. During these review periods, in terms of defecation, the humic ore-dosed groups tended to decrease.

Test Example 6: In vivo test (the effect of oral administration of humic minerals on mice)

Normal diet: ND, High fat diet: HFD, and a mixed feed of HFD and 2% humic ore were administered to mice, and body weight was measured over time.

The result is shown in Figure 8. As can be seen from FIG. 8, weight gain can be suppressed by combining HFD (high-fat diet) with 2% humic mineral. Although not shown in the graph, the intake of feed showed a tendency to increase with the mixed feed of 2% humic mineral. Therefore, the weight change is not the result of mixing humic minerals. Feed will result in lower intake. On the contrary, it will increase the intake by about 2 times, and even if the intake increases, weight loss can still be obtained, so it can be inferred that humic minerals have a metabolism-promoting effect.

HFD, and a mixed feed of HFD and 2% humic mineral were administered to the mice, and the tail was cut to collect blood. Use the Nipro blood glucose self-monitoring device to measure the blood glucose value. The collected blood was subjected to serum separation by a hematocrit tube centrifuge. Use Levis (trademark) insulin-mouse-T (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), Mouse Adiponectin/Acrp30 Quantikine ELISA Kit (R&D systems, Minneapolis, MN) to measure insulin and adiponectin in plasma respectively concentration.

The result is shown in Figure 9. It can be seen from Figure 9 that 0.1% humic mineral can inhibit the rise of blood sugar level in the living body, and it is predicted that it will have a beneficial effect on the pancreas and improve the function of adipose tissue.

For mice administered with ND, HFD and the mixed feed of HFD and 2% humic mineral for 6 weeks, the autopsy results are shown in Fig. 10 . The tissue weight of liver and epididymis increased significantly compared with HFD, and the tissue weight increase of HFD combined with 2% humic mineral was lower than that of HFD (A and C).

The liver and epididymis were fixed with 10% (w/v) paraformaldehyde and embedded in paraffin blocks. The block was sliced into thin slices using a microtome (Leica Biosystems, Wetzlar, Germany). Sections (5 μm thick) were stained with hematoxylin and eosin, and observed using an optical microscope. The results are shown in B and D of Fig. 10 . Any of the fat cell diameters of the liver and epididymis was increased by HFD, but the HFD combined with 2% humic mineral was relatively decreased.

The results of HE staining of adipose tissue of epididymis tissue were determined using the imaging software cellSens (Olympus Co., Ltd.) for microscopy. For each group, tissue samples were obtained from 5 pieces, and 6 sections (6 sections) were stained. 30 cells in one field of view among the five fields of view were counted. The result is shown in Figure 11. Although the fat cell diameter increases with HFD, it will decrease with HFD combined with 2% humic mineral.

Test Example 7: In vivo test (glucose tolerance test in mice)

The results of the glucose tolerance test (OGTT: oral glucose tolerance test) performed on mice are shown in FIG. 12 . In the same manner as above, each of the feeds was fed for 6 weeks, and 100 μl of 300 mg/ml glucose was orally administered, and the blood sugar level was measured over time. As a result, the highest blood sugar level is lower than that of 0.1% humic ore. In addition, after glucose tolerance of 60 points, the decrease of blood sugar level will be improved by 0.1% humic ore. In addition, the same experiment was performed on KK-Ay mice, which are a type II diabetes model, and body weight loss and normal blood sugar levels were reduced by oral administration of humic minerals.

Test Example 8: In vivo test (blood tests related to liver function and lipid metabolism in mice)

Table 1 shows the blood test results of the liver function and lipid metabolism of the mice administered a high-fat diet for 6 weeks. Blood was obtained by sampling cardiac blood, and serum was obtained by centrifugation.

Data represent mean ± S.E.M. n=5

AST, ALT, ALP, LDH, ChE values represent liver function. Compared with the ND group, the liver function of the HFD group deteriorated, but it was confirmed that the liver function could be greatly improved when HFD was mixed with 0.1% humic ore. Compared with the ND group, the neutral fat (TG) and LDL-C values of the HFD group were significantly deteriorated. On the other hand, when HFD was mixed with 0.1% humic mineral, it was confirmed that the lipid metabolism was greatly improved.

Test Example 9: In vivo test (ATP/ADP ratio measurement)

The homogenate of mouse muscle tissue in 1 mL of PBS was centrifuged (4° C., 15,000 rpm, 5 min). For 5 μL of supernatant, luminescence was measured by 20/20n Luminometer using ADP/ATP Assay kit (Sigma-Aldrich, MO, USA). In addition, 15 μL of supernatant was added to 150 μL of working reagent in 1 well of a 96-well plate. After incubating at 37° C. for 15 minutes, the absorbance at 570 nm was measured with a Model 680 Microplate Reader (Bio-Rad Laboratories) using the Protein Assay Bicichoninate Kit (Nacalai Tesque, Kyoto, Japan).

The result is shown in Figure 13. From the ATP/ADP ratio in muscle tissue in Figure 13, it can be known that humic minerals can activate metabolism.

Test Example 10: Effect of Fulvic Acid

The concentration-dependent ATP consumption enhancing effect of fulvic acid was measured by the same method as in Test Example 3. The result is shown in Figure 14. It can be seen from Figure 14 that the effect of pure fulvic acid (provided by the Japanese Society of Humic Substances) can roughly reproduce the effect of humic minerals (fulvic acid + minerals).

In addition, mitochondrial function in C2C12 cells was analyzed using XFe24 flux analyzer Seahorse Bioscience (North Billerica, MA) according to the manufacturer's instructions. Briefly, C2C12 was inoculated onto a dedicated 24-well plate at a concentration of 30,000 cells/well and cultured overnight. In addition, fulvic acid and humic minerals were dissolved in a special medium (hereinafter referred to as the basic medium) with the addition of 1mM pyruvic acid and 2mM glutamine. Carrier means the base medium. At least 40 min before the assay, the medium was exchanged for basic medium. In order to assess OCR by proton leakage, oxygen was monitored in real time by sequential treatment with oligomycin (ATP synthase inhibitor), FCCP (mitochondrial decoupler), and rotenone/antimycin A (respiratory inhibitor). Consumption ratio (OCR, pmol/min). The results are shown in Figures 15 and 16. It can be seen from Figures 15 and 16 that either 0.01% pure fulvic acid or 0.01% humic ore can enhance spare respiration capacity.

From these results, it can be known that fulvic acid and humic minerals can inhibit the synthesis of ATP and indirectly stimulate the transport of sugars through GLUT, not in the aerobic metabolic pathway of mitochondrial respiration, but in the anaerobic metabolic pathway. From this, it can be considered that fulvic acid and humic minerals act as weak decoupling agents, thereby increasing mitochondrial biosynthesis. As a result, it was found that strong metabolism was observed as indicated by the data of ATP/ADP ratio in vivo.

Test Example 11: Analysis Results of Humic Mine

(sample preparation)

Preparation method of the sample: Stir the sample for analysis. Add a small amount of humic mineral powder (hereinafter referred to as the sample) and a small amount of purified water into a 250mL jar, stir evenly to dissolve it. Further, add a small amount of sample and purified water drop by drop, stir evenly to dissolve. Repeat this action to make a 60% (60g/100mL) solution, put it into the rotor, and stir at 20°C for 24 hours using a magnetic stirrer. The solution stirred for 24 hours was transferred to a centrifuge tube, centrifuged (2500 rpm×10 minutes: 2 times), and the resulting supernatant was collected and analyzed as a test solution.

Fractional distillation of fulvic acid: In order to produce alkaline solubles, sodium hydroxide is added to the 60% solution to make it equivalent to a 0.1M-NaOH solution (at this time, 5mL of the 60% solution is used). Use glass fiber filter paper to remove precipitates, and the filtrate is used as alkaline soluble matter. In order to remove the acidic insoluble matter (production of fulvic acid fractionation), add hydrochloric acid to the alkaline soluble matter to make the pH = 2 and let it stand for about 12 hours, then use glass fiber filter paper to remove the acidic insoluble matter. The resulting filtrate was analyzed as a fulvic acid fraction.

Infrared Absorption (FT-IR): Fourier Transform Infrared Spectrophotometry: Samples were dried at 105°C for 2 hours (liquid samples until dry), and analyzed gravimetrically. The dried sample was heated at 600° C. for 2 hours, and analyzed by gravimetric method. Use the purified DAX-8 resin manufactured by Supelco to absorb the resin-adsorbable organic matter in the 4% solution, and then desorb with the alkaline solution, remove the unnecessary salts with the ion-exchange resin, and use the removed one as the analysis sample. The infrared absorption spectrum was obtained by the FT-IR method.

Put the fulvic acid (three-dimensional fluorescence spectrophotometer method) sample into the measurement unit, obtain the three-dimensional excitation-fluorescence spectrum, and perform quantification based on the sum of the fluorescence intensity of the entire wavelength range of the fluorescence expressed by the humic substances. In addition, since the standard material of the fluorescence characteristic is the brown forest soil fulvic acid (promulgated by the Japanese Society of Humic Substances), the obtained value is relative to the fluorescence of the brown forest soil fulvic acid Properties are normalized values. In addition, the relative fluorescence intensity (QSU) was calculated by taking the fluorescence intensity of 10 μg/L quinine sulfate at an excitation wavelength of 345nm and a fluorescence wavelength of 455nm as 10QSU, and calculated the relative fluorescence intensity (QSU) from the sum and peak value of the fluorescence intensity in the above wavelength range. Relative fluorescence intensity (QSU).

(result)

Notes on optical properties: As stated in the analysis method, it is the quantitative value when using the standard substance of fluorescent properties, brown forest soil fulvic acid (promulgated by the Japanese Society of Humic Substances). Therefore, when fulvic acid produced in different places is used as a standard, the quantitative value will be different due to the difference in optical properties (fluorescent properties) due to the difference in molecular structure.

The results are shown in Figures 17 and 18. In addition, Tables 2 and 3 show relative fluorescence intensity (QSU). A peak can be seen near the excitation wavelength of 260nm and the fluorescence wavelength of 440nm. Infrared absorption spectrum (FT-IR) can confirm various functional groups unique to fulvic acid (Figure 17), and three-dimensional fluorescence spectrum shows autofluorescence in the wavelength range unique to fulvic acid (Figure 18). From this, it can be determined that the organic substance contained in this sample is fulvic acid. In addition, compared with the literature value of infrared absorption spectrum, it can be found that compared with the brown forest soil fulvic acid promulgated by the Japanese Society of Humic Substances, it has a tendency to have fewer carboxyl groups and more aromatics.

表中「%」係表示試料濕重量中之含量。

"%" in the table means the content in the wet weight of the sample.

Statistical Analysis

The statistical difference between the two groups was analyzed using Student's t test. A significant difference was judged to exist when the P value was less than 0.05.

Claims (6)

Use of a composition containing fulvic acid and plural minerals, characterized in that it is used in the manufacture of a food composition for the improvement or prevention of lifestyle-related diseases, wherein the food composition does not contain shilajit or marine humus solvent extract. As described in item 1 of the patent application, wherein the aforementioned lifestyle-related diseases are selected from diabetes, insulin resistance, hyperlipidemia, obesity, fatty liver, dyskinesia syndrome, sarcopenia, and cachexia At least 1 species in all groups. The use of a composition comprising fulvic acid and at least one mineral is characterized in that it is used in the manufacture of a food composition for improving liver function. The use of a composition comprising fulvic acid and at least one mineral is characterized in that it is used in the manufacture of an anti-fatigue food composition, wherein the active ingredients of the food composition only contain the fulvic acid and at least one mineral. The use of a composition comprising fulvic acid and at least one mineral is characterized in that it is used in the manufacture of a food composition for muscle enhancement, wherein the active ingredients of the food composition only contain the fulvic acid and at least one mineral. Use of a composition comprising fulvic acid and at least one mineral is characterized in the manufacture of a mitochondrial activator.

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