JP6895309B2 - Composition for promoting AMPK activity - Google Patents

Description

The present invention relates to a composition for promoting AMPK activity.

In modern life, the amount of energy intake tends to exceed the amount of energy consumed, resulting in excess energy due to excessive energy intake due to abundant food and a decrease in energy consumption due to a decrease in the amount of exercise due to the spread of automobiles and the like. For this reason, obesity and associated lifestyle-related diseases are increasing, which has become a major social problem.

One of the measures to improve obesity is to increase the amount of energy consumed by exercising, but especially obese and elderly people have a heavy physical burden, and it is possible to carry out daily exercise. It can be difficult. Therefore, an exercise alternative method that can be expected to have an effect similar to exercise without actually exercising is desired.

AMPK (AMP-activated protein kinase) is a protein that is widely present in living organisms and plays an important role as a regulator of energy metabolism in cells. It is known from previous studies that ATP synthesis is promoted and energy consumption is promoted by increasing the activity of AMPK in a situation where the intracellular ATP level is lowered. In addition, recent studies have shown that substances that activate AMPK are also effective in preventing and improving endurance muscle strength, and not only prevent obesity but also promote health by strengthening muscle mass and strength. It is considered to be useful for.

As a substance having an AMPK activating effect, metformin, which is a therapeutic drug for diabetes, has been reported, but this substance also has a problem of side effects and is difficult to use in foods. Therefore, it is desired that the material has abundant eating experience, is highly safe, and is easily available, but has an AMPK activating effect.

Here, some of the ingredients derived from wakame seaweed, which has been widely eaten since ancient times and has abundant eating experience, are excellent in safety, can be taken and ingested for a long period of time on a daily basis, and are of a certain type. It is known to have physiological activity. For example, a peptide obtained by treating a protein extracted from wakame seaweed with a proteolytic enzyme or the like is known to have an angiotensin converting enzyme inhibitory effect (Patent Documents 1 to 3).

Under such circumstances, it is desirable to provide a novel ingredient derived from wakame seaweed as an active ingredient of the composition for promoting AMPK activity.

Japanese Unexamined Patent Publication No. 2003-128694 JP-A-2002-138100 Japanese Unexamined Patent Publication No. 2001-064299

An object of the present invention is to provide a novel ingredient derived from wakame seaweed as an active ingredient of a composition for promoting AMPK activity.

As a result of diligent studies on the above problems, the present inventor has found an AMPK activity-promoting action in some wakame-derived components, and has come to the present invention based on this finding.

That is, the present invention comprises the following (1) to (3).
(1) An extract obtained by hydrolyzing a wakame that has been subjected to a decomposition treatment of alginic acid, separating and recovering an insoluble fraction of the obtained hydrolyzate, and extracting the recovered insoluble fraction with an organic solvent. A composition for promoting AMPK activity containing.
(2) The composition for promoting AMPK activity according to (1) above, wherein the organic solvent is a hydrocarbon solvent or a lower alcohol.
(3) A composition for promoting AMPK activity containing a peptide derived from wakame seaweed as an active ingredient.

The composition for promoting AMPK activity of the present invention has an AMPK activating action.
The composition for promoting AMPK activity of the present invention has no problem in safety even if it is ingested on a daily basis for a long period of time.

Wakame seaweed as referred to in the present invention is a seaweed taxonomically belonging to the genus Wakame of the family Alaria crassifolia. Types of wakame seaweed include wakame seaweed (Undaria pinnatifida), undaria undarioides, and seaweed (U. peterseniana), all of which are preferably used in the present invention. Further, the wakame seaweed portion is not particularly limited, and any of leaves, bud turnips and the like may be used. The form of wakame seaweed may be, for example, dried wakame seaweed (for example, dried cut wakame seaweed, dried wakame seaweed, etc.), boiled salted wakame seaweed, salted wakame seaweed, raw wakame seaweed, etc. ..

In the extract used in the present invention, at least the wakame that has been subjected to the decomposition treatment of alginic acid is hydrolyzed, the insoluble fraction of the obtained hydrolyzate is separated and recovered, and the recovered insoluble fraction is extracted with an organic solvent. It is obtained by the process of

In the above step, first, alginic acid contained in the raw material wakame seaweed is decomposed. The method for decomposing alginic acid is not particularly limited, but for example, a method of treating wakame seaweed with alginate lyase is preferable. After the decomposition treatment of alginic acid, it is preferable to remove the decomposed alginic acid by centrifuging it, discarding the supernatant, and collecting the precipitate.

Next, the protein contained in the wakame seaweed that has been decomposed with alginic acid is hydrolyzed. The method of hydrolysis is not particularly limited, but it is preferable to add, for example, a proteolytic enzyme to decompose the protein. Examples of the proteolytic enzyme include protease, trypsin, pancreatin, chymotrypsin, pepsin, papain, etc. Among them, protease is preferably used.

When the above-mentioned alginate lyase and proteolytic enzyme are used, the amount of enzyme used, the temperature and pH of the treatment may be as long as they are within the range that is generally accepted, but the optimum amount and temperature of the enzyme are used. It is desirable to carry out at the optimum pH in terms of shortening the reaction time and stabilizing the enzyme.

After hydrolysis, the insoluble fraction of the obtained hydrolyzate is separated and recovered. The method is not particularly limited, and examples thereof include a method in which the hydrolyzate is centrifuged, the supernatant is discarded, and the precipitate is recovered. The recovered insoluble fraction is preferably dried by a method known per se.

Next, the recovered insoluble fraction is extracted with an organic solvent to obtain an extract. As the organic solvent, for example, a hydrocarbon solvent or a lower alcohol is preferably used.

As the hydrocarbon solvent, for example, one or more selected from pentane, hexane, cyclohexane, methylcyclohexane, heptane, octane, isooctane, benzene, toluene, ethylbenzene, propylbenzene, o-xylene, m-xylene and p-xylene. Is mentioned, and hexane is preferable.

Examples of the lower alcohol include one or more selected from methanol, ethanol, propanol and butanol, and ethanol is preferable. In addition, these lower alcohols may contain water (hereinafter, those containing water are referred to as "hydrous lower alcohols"). The lower alcohol concentration (v / v) in the hydrous lower alcohol is preferably 20 to 95% by volume, more preferably 50 to 95% by volume.

The amount of the organic solvent used is preferably 1 to 100 mL with respect to 1 g of the insoluble fraction (in terms of dry mass), and the extraction time is preferably 1 minute to 24 hours, more preferably 10 minutes to 10 hours. The extraction temperature at this time is preferably 0 ° C. to the boiling point of the solvent, more preferably 5 to 80 ° C., further preferably 5 to 50 ° C., and even more preferably 10 to 40 ° C.

The extraction means for extracting the insoluble fraction with an organic solvent is not particularly limited as long as the insoluble fraction is brought into contact with the organic solvent (for example, immersion or the like), but the contact is made from the viewpoint of shortening the extraction time. In addition, means such as ultrasonic irradiation, microwave irradiation, and stirring can be used.

It is preferable that the extract thus obtained is concentrated, dried and pulverized by removing the insoluble fraction of the solvent by a method known per se. More specifically, the supernatant can be collected by centrifugation, concentrated by an evaporator, further dried by spray drying, freeze drying, vacuum drying or the like, and pulverized.

The wakame-derived peptide used in the present invention is obtained by separating the protein separated from wakame seaweed from a processed product obtained by treating it with a proteolytic enzyme.

The method for separating the protein from the wakame seaweed is not particularly limited. For example, the wakame seaweed is treated with alginate lyase, and the seaweed is centrifuged to discard the supernatant and collect the precipitate to remove the viscous polysaccharide. However, there is a method of separating the protein.

In the treatment with a proteolytic enzyme, the proteolytic enzyme is added to the protein separated from the wakame seaweed to decompose the protein. Examples of the proteolytic enzyme include protease, trypsin, pancreatin, chymotrypsin, pepsin, papain, etc. Among them, protease is preferably used.

The amount of enzyme used, the temperature and pH of the treatment by the above-mentioned alginate lyase and proteolytic enzyme may be within the range that is generally accepted, but the optimum amount, optimum temperature and optimum pH of the enzyme are used. It is desirable to do this in terms of shortening the reaction time and stabilizing the enzyme.

The method for separating the peptide from the treated product obtained by the treatment with a proteolytic enzyme is not particularly limited. For example, the treated product is centrifuged, undegraded protein is removed to obtain a supernatant, and then the peptide is obtained. Examples thereof include a method in which the supernatant is treated with an ultrafiltration membrane to obtain a peptide having a molecular weight of 10,000 or less.

In the composition for promoting AMPK activity of the present invention, the above-mentioned wakame-derived ingredient (the above extract or the wakame-derived peptide) can be used as it is as an active ingredient, or a pharmaceutically acceptable additive, food material, or food raw material can be used. , Further, if necessary, food additives and the like are appropriately mixed, and according to a conventional method, for example, as a drug, a non-medicinal product or a food or drink (including a health food or a food for specified health use) for the purpose of promoting AMPK activity. Manufactured. Examples of the drug or quasi-drug include oral or parenteral preparations such as powders, granules, tablets, microcapsules, soft capsules, hard capsules, syrups, elixirs, injections, and infusions. The food or drink can take any food form such as a solid food, a creamy or jam-like semi-solid food, a gel-like food, or a beverage. Specific examples include soft drinks, supplements, cookies, puddings, jelly confectionery, candies, drops, chewing gum, chocolate, yogurt, ice cream, margarine, shortening, mayonnaise or dressings.

Examples of additives, food materials, food raw materials or food additives used in the production of the above-mentioned pharmaceuticals, non-pharmaceutical products or foods and drinks include excipients (lactose, dextrin, corn starch, crystalline cellulose, etc.) and lubricants. (Magnetic acid stearate, sucrose fatty acid ester, glycerin fatty acid ester, etc.), disintegrant (calcium carboxymethyl cellulose, anhydrous calcium hydrogen phosphate, calcium carbonate, etc.), binder (starch paste solution, hydroxypropyl cellulose solution, labia gum solution, etc.) , Dissolving aids (Arabic gum, polysorbate 80, etc.), Sweeteners (sugar, fructose, glucose liquid sugar, honey, aspartame, etc.), Colorants (β-carotene, edible tar pigment, riboflavin, etc.), Preservatives (sorbic acid) , Methyl paraoxybenzoate, sodium sulfite, etc.), thickener (sodium alginate, sodium carboxymethyl cellulose, sodium polyacrylate, etc.), antioxidant (BHT, BHA, ascorbic acid, tocopherol, etc.), fragrance (hakka, strawberry fragrance, etc.) Etc.), acidity agents (citric acid, lactose, DL-apple acid, etc.), seasonings (DL-alanine, 5'-sodium inosinate, sodium L-glutamate, etc.), emulsifiers (glycerin fatty acid ester, sucrose fatty acid ester, etc.) ), Acidity regulators (citric acid, trisodium citrate, etc.), vitamins, minerals, amino acids, etc.

When the composition for promoting AMPK activity of the present invention is used as a pharmaceutical product or a quasi-drug, it is pharmaceutical or food hygienic in addition to the above-mentioned additives, food materials, food raw materials or food additives. Other materials to be prepared may be appropriately added and mixed by a conventional method. Such substances are not particularly limited, and examples thereof include coating agents, absorption promoters, stabilizers, and Chinese herbal medicines (herbal extracts).

Here, AMPK belongs to serine / threonine kinase, and it is known that AMPK is activated by phosphorylation of its 172nd threonine. Activated AMPK phosphorylates major enzymes in glucose metabolism and lipid metabolism, and promotes beta-oxidation of fatty acids and glucose uptake. In muscle tissue, activation of AMPK promotes membrane translocation of GLUT-4, which is a sugar transport carrier, and promotes beta-oxidation of fatty acids, sugar uptake, and the like. Therefore, the composition for promoting AMPK activity of the present invention can be used for lifestyle-related diseases such as obesity, hyperglycemia, hyperlipidemia, diabetes and arteriosclerosis by activating AMPK, and diseases and conditions related thereto. , Can be used for prevention and / or treatment. In addition, the composition for promoting AMPK activity of the present invention can also be used as a lipid metabolism promoter, a sugar metabolism promoter, a fat accumulation inhibitor, an exercise substitute, and the like.

When the composition for promoting AMPK activity of the present invention is orally ingested, the ingestion amount varies depending on the sex, age, etc. of the subject to be ingested. The dry matter equivalent amount of the peptide derived from wakame is in the range of 0.01 to 100 mg / body weight 1 kg, preferably 0.05 to 20 mg / body weight 1 kg. This dose may be taken once a day or in several divided doses. However, the actual dose should be determined in consideration of the purpose and the condition of the ingestor (gender, age, health condition, etc.).

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereto.

[Manufacturing of wakame-derived ingredients (hexane extract)]
To 100 g of dried wakame seaweed, 2 L of 0.1 M citric acid buffer was added to adjust the pH to 7.0, and 100 U of alginate lyase (trade name: alginate lyase S; manufactured by Nagase ChemteX Corporation) was added thereto, and then 45 ° C. Was treated for 4 hours. Then, the obtained processed product was centrifuged at 3000 × g for 5 minutes, and the supernatant was discarded to obtain a precipitate. After suspending and dispersing this precipitate in 1.5 kg of water, 10,000 U of protease (trade name: Protease S "Amano"; manufactured by Amano Enzyme) was added to adjust the pH to 8.0, and then 70 ° C. Was treated for 18 hours. Then, the mixture was centrifuged at 3000 × g for 5 minutes, and the precipitate was collected. The precipitate was dried, and a mixture of 1 g of the obtained dried product to which 10 mL of hexane was added was sonicated for 30 minutes and centrifuged at 3000 × g at 20 ° C. for 10 minutes to recover the supernatant. The supernatant was collected after filtration through cotton wool and concentrated at 40 ° C. and 270 Torr for 1 hour using a rotary evaporator. The obtained concentrate was vacuum-dried at 20 ° C. for 12 hours under the condition of 100 Torr using a vacuum low-temperature dryer (model: DP-41; manufactured by Yamato Scientific Co., Ltd.), and the wakame-derived component (hexane extract) 35. 3 mg was obtained. 2.5 mL of DMSO was added to the obtained extract to prepare a sample for AMPK activity test described later.

[Manufacturing of wakame-derived ingredients (ethanol extract)]
To 100 g of dried wakame seaweed, 2 L of 0.1 M citric acid buffer was added to adjust the pH to 7.0, and 100 U of alginate lyase (trade name: alginate lyase S; manufactured by Nagase ChemteX Corporation) was added thereto, and then 45 ° C. Was treated for 4 hours. Then, the obtained processed product was centrifuged at 3000 × g for 5 minutes, and the supernatant was discarded to obtain a precipitate. After suspending and dispersing this precipitate in 1.5 kg of water, 10,000 U of protease (trade name: Protease S "Amano"; manufactured by Amano Enzyme) was added to adjust the pH to 8.0, and then 70 ° C. Was treated for 18 hours. Then, the mixture was centrifuged at 3000 × g for 5 minutes, and the precipitate was collected. The precipitate was dried, and a mixture of 1 g of the obtained dried product to which 10 mL of 100% ethanol was added was sonicated for 30 minutes and centrifuged at 3000 × g at 20 ° C. for 10 minutes to collect the supernatant. did. The supernatant was collected after filtration through cotton wool, and concentrated for 1 hour at 40 ° C. and 130 Torr using a rotary evaporator. The obtained concentrate was vacuum-dried at 20 ° C. for 12 hours under the condition of 100 Torr using a vacuum low-temperature dryer (model: DP-41; manufactured by Yamato Scientific Co., Ltd.), and 120 mg of wakame-derived component (ethanol extract) was added. Obtained. 4 mL of DMSO was added to the obtained extract to prepare a sample for AMPK activity test described later.

[Manufacture of seaweed-derived peptide]
To 100 g of dried wakame seaweed, 2 L of 0.1 M citric acid buffer was added to adjust the pH to 7.0, and 100 U of alginate lyase (trade name: alginate lyase S; manufactured by Nagase ChemteX Corporation) was added thereto, and then 45 ° C. Was treated for 4 hours. Then, the obtained processed product was centrifuged at 3000 × g for 5 minutes, and the supernatant was discarded to obtain a precipitate. After suspending and dispersing this precipitate in 1.5 kg of water, 10,000 U of protease (trade name: Protease S "Amano"; manufactured by Amano Enzyme) was added to adjust the pH to 8.0, and then 70 ° C. Was treated for 18 hours. Then, the supernatant was collected by centrifugation at 3000 × g for 5 minutes. The collected supernatant is ultrafiltered with an ultrafiltration membrane (trade name: Ultrafree-MC; manufactured by Millipore), and after collecting the permeate, a freeze dryer (model: RLE II-103; Kyowa Vacuum Technology Co., Ltd.) Was freeze-dried at −20 ° C. for 12 hours under the condition of a vacuum degree of 20 Pa to obtain 10 g of a powdered wakame-derived peptide. 1 mL of PBS was added to 10 mg of this wakame-derived peptide to prepare a sample for the AMPK activity test described later.

[AMPK activity test]
Mouse muscle cell line (C2C12) ^{was seeded on a 6-well plate to 1.0 × 10 5} cells / well, and in a culture medium containing 2 mL / well of DMEM (+ 10% FBS, + 1% antibacterial agent) at 37 ° C. The cells were cultured for 3 days under the conditions of 5% CO ^{2/95% air.} Then, the culture solution was replaced with DMEM (+ 2% Calf-Serum, + 1% antibacterial agent), and the cells were cultured while exchanging the culture solution every day for another 5 days. After removing the culture solution, DMEM (-FBS) was added and the cells were further cultured for 24 hours. After removing the medium, the above-mentioned test sample was used to (1) a medium containing a wakame-derived component (hexane extract) at a concentration of 50 μg / mL, and (2) a wakame-derived component (ethanol extract) at a concentration of 50 μg / mL. ), And (3) a medium containing a wakame-derived peptide at a concentration of 100 μg / mL were prepared, added to each well, and cultured for 24 hours. A medium containing only PBS or DMSO used for preparing the test sample was added to each well as a control, and the cells were cultured in the same manner.

After removing the culture medium, the cells were washed with PBS and the cells were collected. 100 μL of RIPA Buffer (manufactured by Thermo Fisher Scientific) was added, and homogenization was performed with an ultrasonic crusher. After centrifugation for 15 minutes under the conditions of 14000 rpm and 4 ° C., the supernatant was collected. The protein concentration of the supernatant was measured by a Pierce 660 nm Protein Assay Regent (trade name: manufactured by Thermo Fisher Scientific), an electrophoresis sample was prepared so that the protein content was 20 μg, and the electrophoresis sample was SDS-. It was subjected to PAGE (10% gel).

After transfer to the PVDF membrane, AMPK antibody (trade name: F6 Mouse mAB Cell; manufactured by Signaling Technology) or P-AMPK antibody (trade name: Thr172 Labbit mAB; Cell Signaling Technology, manufactured by Cell Signaling Technology) is used as the primary antibody. (Product name: Goat antibody-Mouse IRDye 800CW; manufactured by LI-COR) or Anti-pAMPK (trade name: Donkey anti-Labbit IgG Jackson; manufactured by ImmunoResearch Labo) as a secondary antibody, and AMPK or phosphorus oxidized. Was detected. The degree of AMPK activation is determined by quantifying the intensity of the detected band using image analysis software ImageJ (National Institutes of Health; http: //rsbweb.nih.gov/ij), and setting the AMPK phosphoric acidity of the control to 100. And shown as a relative value to it. The results are shown in Table 1.

From the results in Table 1, it can be seen that the wakame-derived component (hexane extract), the wakame-derived component (ethanol extract) and the wakame-derived peptide have a strong AMPK activating effect.

[Explanation of impossible / impractical circumstances regarding product-by-process claims]
Claims 1 and 2 of the present application are so-called product-by-process claims for specifying a product by a manufacturing method, but the invention according to these claims is impossible to specify the structure or property of the product as follows. There are impractical circumstances.
That is, it is known that wakame seaweed, which is a natural product, contains a wide variety of components such as carbohydrates such as alginic acid and fucoidan, which are water-soluble polysaccharides, and inorganic substances such as proteins, lipids, vitamins, and minerals. However, not all of them have been clarified.
The inventions according to claims 1 and 2 of the present application use wakame seaweed as a starting material, and use a composition obtained through decomposition treatment of alginic acid, recovery of an insoluble fraction by hydrolysis treatment, and extraction treatment with an organic solvent as an active ingredient. It is a thing. According to the common general knowledge as of the filing of the present application, it is presumed that the composition obtained by this series of steps contains various kinds of polymer components such as fucoidan and indigestible proteins that are not decomposed by hydrolysis treatment. However, since many of these polymer components have an unknown structure, a method for analyzing these components has not been established at the time of filing the application, and the analysis is extremely difficult. Further, it is also assumed that the above composition contains a trace component in an amount less than the detection limit of the analytical instrument, and the trace component has an AMPK activity promoting action.
From the above, the inventions according to claims 1 and 2 of the present application cannot directly specify the structure or characteristics of the object at the time of filing the application of the present application, or consume significantly excessive economic expenditure and time. Since it is necessary, it is considered that it corresponds to an impractical situation.

Claims (2)

The wakame seaweed that has been hydrolyzed with alginic acid is hydrolyzed, the insoluble fraction of the obtained hydrolyzate is separated and recovered, and the recovered insoluble fraction is extracted with hexane or ethanol , and the obtained extract is effective. A composition for promoting AMPK activity as an ingredient. A composition for promoting AMPK activity containing a peptide derived from wakame seaweed as an active ingredient (excluding those used for improving lipid metabolism and / or preventing obesity) .