WO2022158503A1 - 筋肉量の増加及び低下抑制のための組成物、筋肉量の増加及び筋萎縮を抑制するための組成物 - Google Patents
筋肉量の増加及び低下抑制のための組成物、筋肉量の増加及び筋萎縮を抑制するための組成物 Download PDFInfo
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- WO2022158503A1 WO2022158503A1 PCT/JP2022/001881 JP2022001881W WO2022158503A1 WO 2022158503 A1 WO2022158503 A1 WO 2022158503A1 JP 2022001881 W JP2022001881 W JP 2022001881W WO 2022158503 A1 WO2022158503 A1 WO 2022158503A1
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- spirulina
- phycocyanin
- composition
- muscle
- muscle mass
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/748—Cyanobacteria, i.e. blue-green bacteria or blue-green algae, e.g. spirulina
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
Definitions
- the present invention relates to a composition for increasing muscle mass and suppressing muscle loss, and a composition for increasing muscle mass and suppressing muscle atrophy.
- sarcopenia the age-related decline in muscle strength and muscle mass
- methods to deal with it are attracting attention.
- Sarcopenia is accompanied by a decrease in skeletal muscle mass, but in a broader sense, it is also considered to include skeletal muscle weakness and decreased physical function.
- Sarcopenia is thought to involve various factors such as decreased physical activity that changes with age, as well as nutritional intake, hormones, and inflammatory response. Once affected by sarcopenia, a vicious cycle of easy falls/falls, bone fractures, restriction of body movement, and progression of sarcopenia occurs, leading to bedridden conditions.
- Spirulina is a cyanobacterial species that is rich in proteins, sugars, various vitamins, minerals and vegetable pigments. Spirulina is expected to have many functionalities in itself and substances derived from Spirulina. There are active researches on
- a lipase activity inhibitor containing phycocyanin as an active ingredient has been reported as spirulina-derived phycocyanin has an effect of inhibiting the activity of lipases such as pancreatic lipase (see Patent Document 1).
- a serum lipid-lowering agent containing phycocyanin as an active ingredient has been reported, assuming that spirulina-derived phycocyanin has a higher serum lipid-improving effect than soybean protein (see Patent Document 2).
- oral administration of spirulina spirulina extract
- induces the activation of natural killer (NK) cells see Non-Patent Document 1.
- An object of the present invention is to provide a composition for increasing muscle mass and suppressing muscle mass loss, and a composition for suppressing muscle mass increase and muscle atrophy, which can be orally ingested continuously on a daily basis.
- a composition for increasing muscle mass and inhibiting its loss containing as an active ingredient at least one selected from the group consisting of spirulina, phycocyanin, an enzymatic degradation product of spirulina and an enzymatic degradation product of phycocyanin.
- the enzyme in the spirulina enzymatic degradation product and the phycocyanin enzymatic degradation product is a protease.
- a composition for suppressing increase in muscle mass and muscle atrophy containing as an active ingredient at least one selected from the group consisting of spirulina, phycocyanin, enzymatic degradation products of spirulina and enzymatic degradation products of phycocyanin.
- composition according to [5] which is a health food, functional food, dietary supplement, supplement, food with health claims, food for specified health uses, nutritional food, food with function claims, or food for patients.
- composition of [7] which is a pharmaceutical composition used for treating, preventing, or improving sarcopenia or locomotive syndrome.
- compositions for increasing muscle mass and suppressing muscle loss and a composition for increasing muscle mass and suppressing muscle atrophy, which can be orally ingested continuously on a daily basis. can be done.
- FIG. 1 is a graph showing the ratio of expression levels of genes assumed to be involved in the increase and suppression of decrease in muscle mass in the gastrocnemius muscle to the A1 group (Sham group) in Test 1.
- FIG. 1 is a diagram showing the ratio of expression levels of genes assumed to be involved in muscle atrophy in the gastrocnemius muscle to the A1 group (Sham group) in Test 1.
- FIG. 1 is a diagram showing the ratio of expression levels of genes assumed to be involved in muscle atrophy in the gastrocnemius muscle to the A1 group (Sham group) in Test 1.
- the composition according to the present embodiment contains, as an active ingredient, at least one selected from the group consisting of spirulina, phycocyanin, enzymatic degradation products of spirulina, and enzymatic degradation products of phycocyanin, a composition for increasing muscle mass and suppressing loss. about things.
- the composition according to the present embodiment contains, as an active ingredient, at least one selected from the group consisting of spirulina, phycocyanin, enzymatic degradation products of spirulina, and enzymatic degradation products of phycocyanin, and inhibits muscle mass increase and muscle atrophy. It relates to a composition for Hereinafter, after explaining the active ingredients of the composition according to the present embodiment, the application, form, etc. of the composition will be explained.
- Spirulina is one of the active ingredients in the composition according to the present embodiment, and has the effects of increasing muscle mass, suppressing loss of muscle mass, and suppressing muscle atrophy.
- Spirulina is a fine spiral alga belonging to the genus Spirulina of the genus Spirulina of the family Nostrils, family of Cyanophyta, and contains abundant proteins, sugars, various vitamins, minerals, and vegetable pigments.
- Spirulina examples include, for example, Spirulina platensis, Spirulina maxima, Spirulina geitleri, Spirulina siamese, Spirulina subspira meyer Spirulina subsalasa), Spirulina princeps, Spirulina laxissima, Spirulina curta, Spirulina spirulinoides, and the like.
- Spirulina that is easily available and preferable because it can be cultured artificially includes Spirulina platensis, Spirulina geitleri, Spirulina siamese, and the like.
- Spirulina is also called Arthrospira.
- the Spirulina used in the composition according to the present embodiment may be the alga body (wet alga body) cultured in a liquid medium as it is, but the wet alga body is extracted with a solvent such as water or ethanol. It is preferable to use a Spirulina extract, which is an extract obtained by concentrating or drying the extract, or an extract obtained by concentrating or drying the extract.
- the extraction liquid used for producing the Spirulina extract is not particularly limited as long as the effects of the present invention can be obtained, but for example, hot water can be used.
- an extract obtained by hot water extraction of algal spirulina, or an extract obtained by concentrating or drying the extract can be preferably used.
- the method for obtaining Spirulina extract is not particularly limited, and it can be obtained according to a conventional method. can be done. Specifically, the manufacturing method described below can be mentioned.
- Spirulina extract is obtained by extracting Spirulina algae with hot water at a temperature exceeding 100° C., adjusting the pH of the extract under specific acidic conditions, and then removing the insoluble fraction to obtain a Spirulina extract. can be manufactured.
- the Spirulina extract is used in liquid form, the Spirulina extract obtained as described above can be used as it is.
- the Spirulina extract can be concentrated or dried and used as a powder.
- the Spirulina extract used in the composition according to the present embodiment may be a Spirulina extract or one produced by concentrating or drying a Spirulina extract.
- Spirulina for producing the spirulina extract may be commercially available or self-cultivated.
- raw spirulina may be used, or raw spirulina may be dried.
- As a culture method for culturing Spirulina it can be carried out according to a normal method used for culturing blue-green algae.
- Spirulina can be cultured and grown outdoors under basic conditions.
- the cultured Spirulina (hereinafter also referred to as Spirulina algae) can be used as it is, or the cultured Spirulina can be collected with a filter cloth or filter paper, washed with water, and then suspended in water to form a suspension. good.
- a wet alga body obtained by concentrating a culture solution or a suspension may be used, or the wet alga body may be dried by freeze-drying or spray drying (spray drying). It can be powdered.
- Spirulina alga bodies used for hot water extraction may be wet alga bodies, freeze-dried algal bodies, spray-dried algal bodies, crushed algal bodies, or the like.
- the alga bodies are crushed by a conventional method, for example, industrially, by a high-pressure extrusion method such as a French press.
- the Spirulina algal body processed as described above is previously suspended in an extraction solvent such as distilled water in a pressurized container.
- the extraction solvent may be tap water, but distilled water is preferable considering that the extract is applied as a food material.
- the extraction temperature is generally above 100°C, preferably 105°C to 140°C, more preferably 110°C to 130°C.
- the pressure for extraction is preferably 1.0 to 2.5 atmospheres.
- the stirring operation may or may not be performed during extraction, but it is preferable to perform the stirring operation from the viewpoint of thermal efficiency. The longer the extraction time, the greater the amount of extraction, but considering the efficiency, the extraction time is usually preferably 0.5 to 4 hours.
- an operation for removing for example, an operation such as centrifugation or filtration of the suspension may be performed, and a supernatant is obtained by this operation.
- the protein can be aggregated, and the aggregated protein can be separated by centrifugation, filtration, or the like to obtain the Spirulina extract.
- the alga body residue and aggregated protein are similarly removed. Separation may be performed by centrifugation, filtration, or the like.
- the acidic conditions below the isoelectric point of the protein are preferably pH 4.5 or less, more preferably pH 3.75 to 4.25 because protein aggregation and precipitation are maximized, and pH 4.0 is even more preferable.
- Sulfuric acid or hydrochloric acid may be used as the acid to be added when adjusting the pH, but in consideration of practical work processes and use as food materials, it is preferable to use organic acids such as citric acid and malic acid rather than inorganic acids. is preferred.
- Phycocyanin is one of the active ingredients in the composition according to the present embodiment, and has the effects of increasing muscle mass, suppressing muscle loss, and suppressing muscle atrophy.
- Phycocyanin is a chromoprotein and has phycocyanobilin as a chromophore.
- Phycocyanin has a structure in which phycocyanobilin and protein are bound together.
- phycocyanin used in the composition according to the present embodiment examples include phycocyanin derived from algae, such as phycocyanin derived from cyanobacteria, phycocyanin derived from red algae, and phycocyanin derived from cryptophytes. Among these, phycocyanin derived from cyanobacteria is preferable because it can be collected in large amounts.
- blue-green algae examples include the genus Spirulina, the genus Arthrospira, the genus Aphanizomenon, the genus Fisherella, the genus Anabaena, the genus Nostoc, the genus Synechocystis, and the genus Synechocystis. Synechococcus genus, Tolypothrix genus, Aphanothece genus, Mastigoclaus genus, Pleurocapsa genus, and other blue-green algae.
- cyanobacteria belonging to the genus Spirulina and genus Arthrospira which have been produced on an industrial scale and whose safety has been confirmed, are preferred, and cyanobacteria belonging to the genus Spirulina are more preferred.
- raw cyanobacteria may be used, and dried cyanobacteria may be used.
- dried cyanobacteria may be used.
- the dried product of blue-green algae raw blue-green algae may be dried according to a conventional method, or commercially available dried products may be used.
- Phycocyanin includes, for example, C-phycocyanin, R-phycocyanin, allophycocyanin, and the like. From the viewpoint of quality, safety, availability, etc., it is preferable to contain C-phycocyanin as phycocyanin. Therefore, a preferred embodiment of the composition according to this embodiment includes a composition containing C-phycocyanin as an active ingredient. Furthermore, a preferred embodiment of the composition according to this embodiment includes a composition containing C-phycocyanin and allophycocyanin.
- the composition may contain a mixture of phycocyanins consisting of C-phycocyanin and allophycocyanin obtained by extraction from blue-green algae of the genus Spirulina.
- Phycocyanin can be obtained, for example, by suspending blue-green algae in water or a buffer solution such as a phosphate buffer or a citrate buffer and extracting phycocyanin in the blue-green algae.
- a method for extracting phycocyanin is not particularly limited, and it can be extracted according to a conventional method.
- Preferred embodiments of the extraction method include, for example, the extraction method described in JP-A-2006-230272. Specifically, the extraction method described in the following extraction method (i) can be mentioned. By the following extraction method (i), phycocyanin with high purity and bright color tone can be obtained.
- the extraction method (i) is A first step of obtaining an extract obtained by extracting phycocyanin in blue-green algae into an aqueous suspension; a second step of reacting a calcium salt and a phosphate in the extract to produce calcium phosphate and adsorbing contaminants of phycocyanin to the calcium phosphate to obtain an adsorbate; and a third step of removing residues and adsorbates of cyanobacteria from the extract.
- the extraction method (i) is the following extraction method (ii).
- extraction method (ii) of phycocyanin>> The extraction method (ii) is A first step of obtaining an extract obtained by extracting phycocyanin in blue-green algae into an aqueous suspension; a second step of reacting a calcium salt and a phosphate in the extract to produce calcium phosphate and adsorbing contaminants of phycocyanin to the calcium phosphate to obtain an adsorbate; a third step of removing residues and adsorbates of cyanobacteria from the extract; A step of adding a chelating agent to the extract prior to the third step.
- phycocyanin of good quality can be extracted from cyanobacteria.
- the extraction method (i) or (ii) above for blue-green algae of the genus Spirulina it is possible to extract high-quality phycocyanin with a good mixing ratio of C-phycocyanin and allophycocyanin.
- the mixing ratio of C-phycocyanin and allophycocyanin may be adjusted to a desired range by appropriately selecting extraction conditions.
- All types of phycocyanin may be C-phycocyanin.
- allophycocyanin may be included and a mixture of C-phycocyanin and allophycocyanin may be included in the composition.
- the mixing ratio of C-phycocyanin and allophycocyanin is, for example, preferably 3 to 9.5:0.5 to 7, more preferably 6 to 9.5:0.5 to 4, and 7 to 8: 2 to 3 are more preferred.
- the Spirulina enzymatic hydrolyzate is one of the active ingredients in the composition according to the present embodiment, and has the effects of increasing muscle mass, suppressing muscle mass loss, and suppressing muscle atrophy.
- a Spirulina enzymatic degradation product is an enzymatic degradation product obtained by causing an enzyme to act to decompose the above-mentioned Spirulina proteins, sugars, and the like.
- Enzymes used for enzymatic decomposition are not particularly limited, and for example, glucanase, chitinase, protease, pectinase, lipase, cellulase, xylanase, mannanase, hemicellulase, nuclease and the like can be used.
- protease is preferable as the enzyme used for enzymatic decomposition from the viewpoint of suitable decomposition of Spirulina.
- the above enzymes can be used singly or in combination of two or more.
- the origin of the enzyme used for enzymatic decomposition is not particularly limited, and for example, Aspergillus niger, Aspergillus melleus, Aspergillus oryzae, Rhizopus niveus, Bacillus subtilis, Arthrobacter sp., Trichoderma viride and the like can be used.
- protease is not particularly limited, and commercially available protease preparations can be used.
- protease preparations include Sumiteam LP, Sumiteam FL-G, Sumiteam CP, Sumiteam FP-G, Sumiteam MP (Shin Nihon Chemical Industry Co., Ltd.), Bromelain F, Protease P "Amano" 3SD, Papain W-40, Samoase.
- PC10F Samoase C100, Samoase C160, Protin SD-NY10 (Protin SD-PC10F) (Amano Enzyme Co., Ltd.) and the like can be used.
- the ratio of components having a molecular weight of less than 500 is preferably 20% by mass or more, more preferably 30% by mass or more, and even more preferably 35% by mass or more.
- the protein molecular weight distribution of the enzymatic hydrolyzate of Spirulina is at least the above lower limit, the enzymatic hydrolyzate of Spirulina is excellent in bioabsorbability.
- Molecular weight distribution values can be obtained by liquid chromatography analysis of a sample using a gel filtration column.
- the enzymatic decomposition treatment method is not particularly limited, and can be treated according to the usual method.
- the reaction conditions for enzymatic decomposition are not particularly limited, and reaction conditions such as optimum temperature, optimum pH and reaction time may be appropriately adjusted according to the type, activity and amount of enzyme added.
- the Spirulina enzymatic degradation product obtained by the enzymatic degradation treatment is optionally subjected to operations such as centrifugation, filtration, desalting, concentration, drying, solvent extraction, dilution, and addition of additives to obtain the desired form and properties. can be adjusted to
- the Spirulina aqueous solution is heated at 20-70°C. After adjusting the optimum pH of the enzyme using an aqueous solution, 0.01 mass % or more, preferably 0.1 to 10 mass % of the enzyme is added to the protein in Spirulina, and the mixture is stirred for 1 to 24 hours. After stirring, the mixture is heated or cooled to stop deactivation and enzymatic decomposition, and then centrifuged. After centrifugation, the resulting supernatant is freeze-dried to obtain a Spirulina enzymatic hydrolyzate.
- a phycocyanin enzymatic degradation product is one of the active ingredients in the composition according to the present embodiment, and has the effects of increasing muscle mass, suppressing muscle mass loss, and suppressing muscle atrophy.
- a phycocyanin enzymatic degradation product is an enzymatic degradation product obtained by causing an enzyme to act to degrade the phycocyanin described above.
- the enzymes used for enzymatic decomposition are not particularly limited, and the enzymes described in the above Spirulina enzymatic decomposition product can be used, so the description is omitted.
- an enzyme used for enzymatic decomposition it is preferable to use a protease similarly to the Spirulina enzymatic decomposition product mentioned above.
- the proportion of components having a molecular weight of less than 500 is preferably 20% by mass or more, more preferably 30% by mass or more, and even more preferably 35% by mass or more.
- the molecular weight distribution of the phycocyanin enzymatic degradation product is at least the above lower limit, the phycocyanin enzymatic degradation product is excellent in bioabsorbability.
- Molecular weight distribution values can be obtained by liquid chromatography analysis of a sample using a gel filtration column.
- protease used for enzymatic decomposition and the enzymatic decomposition treatment method are not particularly limited, and are the same as those described for the Spirulina enzymatic decomposition product described above, so the description is omitted.
- the phycocyanin aqueous solution is heated at 20-70°C. After adjusting the optimum pH of the enzyme using an aqueous solution, 0.01% by mass or more, preferably 0.1 to 10% by mass of the enzyme is added to the phycocyanin, and the mixture is stirred for 1 to 24 hours. After stirring, the mixture is heated or cooled to stop deactivation and enzymatic decomposition, and then centrifuged. After centrifugation, the resulting supernatant is freeze-dried to obtain a phycocyanin enzymatic hydrolyzate.
- composition for increasing muscle mass and suppressing decrease As shown in the examples below, active ingredients of spirulina, phycocyanin, spirulina enzymatic hydrolyzate, and phycocyanin enzymatic hydrolyzate are found to alleviate the suppression of the expression of genes assumed to be involved in the suppression of muscle mass increase and decrease. was taken.
- genes assumed to be involved in suppressing the increase and decrease in muscle mass whose expression suppression is alleviated by the active ingredient include, for example, Myh13 (Myosin, heavy chain 13), Myh2 (Myosin, heavy chain 2), Myl2 (Myosin , light polypeptide 2), Myh6 (Myosin, heavy chain 6), Tnni1 (Troponin I type 1), Tnnc1 ((Troponin C type 1), Itm2a (Integral membrane protein 2A) and Neu2 (Neuraminidase 2).
- the composition according to the present embodiment contains, as an active ingredient, at least one selected from the group consisting of spirulina, phycocyanin, enzymatic degradation products of spirulina, and enzymatic degradation products of phycocyanin, for increasing and suppressing decrease in muscle mass.
- at least one selected from the group consisting of spirulina, phycocyanin, enzymatic degradation products of spirulina, and enzymatic degradation products of phycocyanin for increasing and suppressing decrease in muscle mass.
- Skeletal muscle is preferred, though not particularly limited, as the target muscle for increasing or suppressing decrease in muscle mass.
- Skeletal muscles are considered to be involved in knee extension and hip joint flexion, specifically, standing up with knee extension, walking, running, and the like.
- Increased skeletal muscle mass improves movements such as standing up, walking, and running, and is particularly effective in the treatment, prevention, or improvement of sarcopenia and locomotive syndrome.
- the locomotive syndrome refers to a symptom of a state in which daily life becomes difficult due to damage to locomotive organs such as muscles and bones due to aging or lack of exercise. While locomotive syndrome is a concept that includes symptoms of locomotive organs in general, sarcopenia is considered to be a symptom focusing on muscle mass, muscle strength, and physical function among locomotive organs.
- Skeletal muscles include, for example, sternocleidomastoid, pectoralis major, pectoralis minor, serratus anterior, subclavius, rectus abdominis, external oblique, internal oblique, transversus abdominis, quadratus lumborum, and caps, latissimus dorsi, erector spinae, levator scapula, rhomboids, deltoids, teres minor, supraspinatus, infraspinatus, subscapularis, teres major, coracobrachialis, biceps , brachialis, brachioradialis, triceps, elbow, pronator teres, pronator quadratus, supinator, flexor carpi ulnaris, flexor carpi radialis, palmaris longus, flexor digiti superficialis, deep flexor digitorum longus exten
- composition for suppressing increase in muscle mass and muscle atrophy As shown in the examples below, active ingredients of spirulina, phycocyanin, enzymatic hydrolyzate of spirulina, and enzymatic hydrolyzate of phycocyanin were found to alleviate the increased expression of genes assumed to be involved in muscle atrophy.
- genes assumed to be involved in muscle atrophy whose expression is suppressed by the above active ingredients include Ddit4 (DNA-damage-inducible transcript 4), Junb (Jun B proto-oncogene), Egr1 (zif-268, Early growth response protein 1, Sdc4 (Syndecan 4), Kcnk5 (Potassium channel, subfamily K, member 5) and Rasd2 (Rhes, RASD family, member 2).
- the composition according to the present embodiment contains as an active ingredient at least one selected from the group consisting of spirulina, phycocyanin, enzymatic degradation products of spirulina, and enzymatic degradation products of phycocyanin, and inhibits muscle mass increase and muscle atrophy. It can be used as a composition for
- Muscular atrophy refers to the loss of muscle mass due to the reduction or shrinkage of muscle cells. (also referred to as aging). Therefore, suppression of muscle atrophy means suppression of loss of muscle mass due to inactivity or aging.
- the target muscles for increasing muscle mass and suppressing muscle atrophy are the same as the muscles described in the composition for increasing and suppressing decrease in muscle mass described above, so a description thereof will be omitted.
- the composition according to the present embodiment can exhibit the action by adding an effective amount of an active ingredient that can effectively exhibit the desired action to various foods as a food material, in addition to general foods. It can be provided as a food composition having.
- the composition according to the present embodiment includes, for example, health food, functional food, dietary supplement, supplement, food with health claims, food for specified health use, food with nutrient function claims, food with function claims, food for the sick, food additive It can be suitably used in food compositions such as agents, feeds and feed additives.
- the form of the food composition is not particularly limited and can be appropriately selected depending on the intended purpose. For example, it may be solid, liquid or gel.
- Foods with function claims are foods labeled with functionalities based on scientific evidence under the responsibility of the business operator. It is a food that has been prepared.
- a food composition for the purpose of increasing and suppressing decrease in muscle mass and / or increasing muscle mass and suppressing muscle atrophy "necessary for leading an independent daily life Helps prevent loss of muscle mass and strength", “Helps improve walking ability”, “Maintains muscle mass and strength”, “Supports maintenance of muscle mass and strength”, “Walking that declines with age in middle-aged and elderly people” It may be labeled as "maintaining ability” or “supporting the ability to build muscle that helps maintain muscle that weakens with age”.
- Examples of food compositions include soft drinks, carbonated drinks, fruit juice-containing beverages, vegetable juice-containing beverages, fruit and vegetable juice-containing beverages, livestock milk such as milk, soy milk, milk beverages, drink-type yogurt, drink-type and Stick-type jelly, coffee, cocoa, tea drinks, nutritional drinks, energy drinks, sports drinks, mineral water, near water, non-alcoholic beverages such as non-alcoholic beer-taste beverages; rice, noodles, bread and pasta Carbohydrate-containing foods and drinks; Cheeses, hard or soft yogurt, dairy products such as fresh cream and ice cream made from other fats and oils; Western confectionery such as cookies, cakes and chocolates; tablets (soft snacks) such as ramune, candies, gums, frozen desserts such as jellies and puddings, frozen desserts, snacks, etc.; whiskey, bourbon, spirits, liqueurs, wine, fruit wine, sake, China Alcoholic beverages such as sake, shochu, beer, non-alcoholic beer with an alcohol content of 1% or less, low-malt beer, miscellaneous alcoholic beverages, and
- food compositions include, for example, health foods, functional foods, dietary supplements, supplements, foods with health claims, foods with specified health uses, foods with nutritional functions, foods with functional claims, foods for the sick, food additives, Feeds and feed additives may be tablets (including chewable tablets, etc.), capsules, troches, syrups, jelly, granules, powders, and the like.
- one or more ingredients that can be used in normal food compositions may be freely selected and blended.
- all additives that can be commonly used in the food field such as various seasonings, preservatives, emulsifiers, stabilizers, fragrances, coloring agents, preservatives and pH adjusters, can be contained.
- composition can be used in the field of pharmaceuticals by blending a pharmaceutically acceptable carrier, an additive, etc. with an effective amount of an active ingredient that can effectively exhibit the desired action. It can be provided as a pharmaceutical composition having The composition according to this embodiment can be suitably used, for example, as a pharmaceutical composition for treating, preventing, or improving sarcopenia or locomotive syndrome described above.
- the pharmaceutical composition may be a pharmaceutical or a quasi-drug.
- the form of the pharmaceutical composition is not particularly limited and can be appropriately selected depending on the intended purpose. For example, it may be solid, liquid or gel.
- the pharmaceutical composition contains conventional pharmaceutically acceptable carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, solubilizers, solubilizers, tonicity agents and the like. Additives and the like can be contained.
- the pharmaceutical composition may be for oral or parenteral use, but is more preferably for oral use. For oral use, commonly used dosage forms such as tablets, powders, granules, capsules, syrups and suspensions can be used.
- dosage forms for example, injections (subcutaneous injection, intravenous injection, intramuscular injection, etc.) in dosage forms such as solutions, emulsions, suspensions, etc., or intranasal injections in spray dosage forms administration and the like.
- the active ingredient is added with excipients, binders, disintegrants, lubricants, preservatives, antioxidants, It can be prepared according to a conventional method by appropriately combining additives such as tonicity agents, buffering agents, coating agents, flavoring agents, solubilizing agents, bases, dispersing agents, stabilizers and coloring agents.
- excipients examples include starch and its derivatives (dextrin, carboxymethyl starch, etc.), cellulose and its derivatives (methylcellulose, hydroxypropyl methylcellulose, etc.), sugars (lactose, white sugar, glucose, trehalose, etc.), citric acid or its salts. , malic acid or its salts, ethylenediaminetetraacetic acid or its salts.
- Binders include starch and derivatives thereof (pregelatinized starch, dextrin, etc.), cellulose and derivatives thereof (ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, etc.), gum arabic, tragacanth, gelatin, sugars (glucose, sucrose, etc.), Ethanol etc. are mentioned.
- Disintegrants include starch and derivatives thereof (carboxymethyl starch, hydroxypropyl starch, etc.), cellulose and derivatives thereof (sodium carboxymethyl cellulose, crystalline cellulose, hydroxypropylmethyl cellulose, etc.), carbonates (calcium carbonate, calcium hydrogen carbonate, etc.), Tragacanth, gelatin, agar and the like.
- Lubricants include stearic acid, calcium stearate, magnesium stearate, talc, titanium oxide, calcium hydrogen phosphate, dried aluminum hydroxide gel, sucrose fatty acid esters, and edible oils and fats.
- Preservatives include paraoxybenzoic acid esters, sulfites (sodium sulfite, sodium pyrosulfite, etc.), phosphates (sodium phosphate, calcium polyphosphate, sodium polyphosphate, sodium metaphosphate, etc.), dehydroacetic acid, sodium dehydroacetate , glycerin sorbate, sugars, and the like.
- Antioxidants include sulfites (sodium sulfite, sodium hydrogen sulfite, etc.), erythorbic acid, L-ascorbic acid, cysteine, thioglycerol, butylhydroxyanisole, dibutylhydroxytoluene, propyl gallate, ascorbyl palmitate, dl- Examples include ⁇ -tocopherol.
- Tonicity agents include sodium chloride, sodium nitrate, potassium nitrate, dextrin, glycerin, glucose and the like.
- buffering agents include sodium carbonate, hydrochloric acid, boric acid, phosphates (sodium hydrogen phosphate, etc.), and the like.
- Coating agents include cellulose derivatives (hydroxypropylcellulose, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, etc.), shellac, polyvinylpyrrolidone, polyvinylpyridines (poly-2-vinylpyridine, poly-2-vinyl-5-ethylpyridine etc.), polyvinyl acetyldiethylaminoacetate, polyvinyl alcohol phthalate, methacrylate/methacrylic acid copolymer, and the like.
- flavoring agents include sugars (glucose, white sugar, lactose, etc.), saccharin sodium, sugar alcohols, and the like.
- solubilizers include ethylenediamine, nicotinamide, saccharin sodium, citric acid, citrates, sodium benzoate, polyvinylpyrrolidone, polysorbates, sorbitan fatty acid esters, glycerin, polypropylene glycol, and benzyl alcohol.
- Bases include fats (lard, etc.), vegetable oils (olive oil, sesame oil, etc.), animal oils, lanolinic acid, petroleum jelly, paraffin, resins, bentonite, glycerin, glycol oils, and the like.
- Dispersants include gum arabic, tragacanth, cellulose derivatives (methylcellulose, etc.), sodium alginate, polysorbates, sorbitan fatty acid esters, and the like.
- stabilizers include sulfites (sodium hydrogen sulfite, etc.), nitrogen, carbon dioxide, and the like.
- the total content of active ingredients in the food composition and / or pharmaceutical composition according to the present embodiment is the type of food or drug, the ingredient , form, etc., and is not particularly limited as long as the effects of the present invention can be obtained, and can be selected as appropriate.
- the total content of active ingredients in the tablet is particularly limited within the range where the effects of the present invention can be obtained. However, it is preferably 20% by mass or more, more preferably 50% by mass or more, in terms of the dry weight of the active ingredient in the total mass of the food composition and/or pharmaceutical composition. Moreover, the total content may be 100% by mass or less, preferably 99% by mass or less.
- the total intake of active ingredients is not particularly limited, and is appropriately selected depending on the type and ingredients of foods and medicines. 0.01 g or more is preferable, 0.03 g or more is more preferable, 10 g or less is preferable, and 4 g or less is more preferable.
- Spirulina platensis was grown under basic conditions (pH 11) in outdoor ponds.
- 50 g of the grown Spirulina platensis spray-dried algal body powder was suspended in 500 mL of distilled water in an autoclave and extracted at an extraction temperature of 120° C. for 1 hour by adjusting the pressure.
- the pH of the extract was adjusted to 4.0 with citric acid. This was centrifuged to remove the alga body residue and protein (insoluble fraction) to obtain a Spirulina extract, which is a Spirulina hot water extract. After the obtained spirulina extract was spray-dried, it was crushed to obtain powdery spirulina.
- ⁇ Phycocyanin>> Add 65 kg of Spirulina dry algal body (spray-dried product) produced in an outdoor culture tank to 1300 L of a 1% calcium chloride (anhydrous) solution, stir for 15 minutes to form a uniform suspension, and then stand at 20° C. for 15 hours. Phycocyanin in the blue-green algae was extracted into the solution below to obtain an extract. 32 kg of sodium dihydrogen phosphate was added to this extract, and after stirring for 0.5 hour, the reaction was allowed to stand at 20° C. for 2.5 hours to generate calcium phosphate and adsorb phycocyanin contaminants to the calcium phosphate. to obtain the adsorbate.
- the extract was introduced into a centrifuge and centrifuged at a gravitational acceleration of 10,000 G for 15 minutes to remove cyanobacterial residues and adsorbates from the extract.
- the resulting phycocyanin extract is subjected to ultrafiltration using a separation membrane with a cutoff molecular weight of 10,000 to remove low molecular weight components and salts, then trehalose and trisodium citrate are added and mixed, followed by spray drying. 15 kg of dried phycocyanin pigment was obtained. This was designated as phycocyanin.
- the phycocyanin content was about 30% by mass (about 22% by mass of C-phycocyanin and about 8% by mass of allophycocyanin) in 100% by mass of the phycocyanin pigment powder.
- protin SD-NY10, manufactured by Amano Enzyme Co., Ltd.
- reaction solution After the reaction solution was cooled to room temperature, it was led to a centrifuge and centrifuged at a gravitational acceleration of 10,000 G for 15 minutes. After collecting the supernatant, it was freeze-dried to obtain 16 g of a phycocyanin enzymatic degradation product, which is a protease degradation product of phycocyanin.
- mice ⁇ Test system> Experimental groups, test substances, doses, treatments and number of cases are shown in Table 1 below. As for the frequency and period of administration, the drug was orally administered once a day for 21 days (administration liquid volume: 10 mL/kg). Group A1 is also referred to as a Sham group (normal rat group without sciatic nerve resection), and Group A2 is also referred to as a vehicle group.
- the average muscle mass of each muscle in the A3-A6 groups was compared with the A2 group (vehicle group), and the rate of increase (%) in muscle mass in the A3-A6 groups was calculated (each Average muscle mass (% by weight) of muscle/average muscle mass (% by weight) of each muscle in both hindlimbs of group A2 (vehicle group) ⁇ 100). Furthermore, the rate of change of each muscle in groups A1 to A6 (average wet weight of each muscle of left hind limb (g)/average wet weight of each muscle of right hind limb (g) ⁇ 100, weight %) was calculated.
- RNA was extracted from the resected gastrocnemius muscle of each of the A1 to A3 and A6 groups and used for DNA microarray.
- DNA microarray was performed as follows.
- RNA samples were pooled for each group, and cDNA synthesis, Cy3-labeled cRNA synthesis and purification were performed using the Low Input Quick Amp Labeling Kit (Agilent).
- concentration of the resulting labeled cRNA and Cy3 incorporation were calculated from the absorbance at 260 nm, 280 nm, 550 nm and 320 nm, and the standard value (Cy3-CTP incorporation > 6 pmol/ ⁇ g) was confirmed to be satisfied.
- each labeled cRNA was fragmented, applied to Whole Mouse Genome Microarray Ver2.0 (Agilent), and hybridized at 65°C for 17 hours.
- Array slides were then washed using Gene Expression Wash Buffers 1 and 2 (Agilent). Array images scanned with a microarray scanner were digitized with array analysis software GenePix Pro (Molecular Devices). Fluorescence intensity values were normalized, and ratios of other groups to the Sham group were calculated.
- Table 2 shows the results of muscle mass increase rate.
- the muscle mass increase rate in the A6 group to which phycocyanin was administered was 102.0%, and the muscle mass increased.
- the increase rate of muscle mass in group A4 to which the enzymatic hydrolyzate of spirulina was administered was 103.7%, and the muscle mass increased.
- the increase rate of muscle mass in the A5 group to which the phycocyanin enzymatic hydrolyzate was administered was 102.5%, and the muscle mass increased.
- the muscle mass increase rate of the A6 group to which phycocyanin was administered was 105.0%, and the muscle mass increased.
- Table 3 shows the results of the rate of change.
- the rate of change in the gastrocnemius muscle and soleus muscle of group A2 was significantly reduced compared to group A1 (sham group), which is a normal rat.
- group A1 sham group
- the rate of change in group A4 administered with the spirulina enzymatic hydrolyzate and in group A6 administered with phycocyanin increased compared to group A2.
- FIG. 1 shows the results of gene expression levels assumed to be involved in the increase and suppression of muscle mass decrease.
- FIG. 1 is a diagram showing the ratio of expression levels of genes assumed to be involved in the increase in muscle mass in the gastrocnemius muscle and suppression of decrease in muscle mass in the A1 group (Sham group). As shown in Figure 1, the administration of spirulina or phycocyanin in the gastrocnemius muscle significantly affected the production of muscle structural proteins Myh13, Myh2, Myl2, Myh6 and troponin, which are related to myosin production, compared with the A2 group (vehicle group).
- FIG. 2 shows the results of muscle atrophy-related gene expression levels.
- FIG. 2 is a graph showing the ratio of expression levels of genes assumed to be involved in muscle atrophy in the gastrocnemius muscle in the A1 group (Sham group).
- administration of spirulina or phycocyanin in the gastrocnemius muscle showed that Ddit4, Junb, and Egr1 involved in muscle atrophy and muscle regeneration, and Sdc4, Kcnk5 and Sdc4 involved in muscle regeneration, compared with the A2 group (vehicle group). It was found that the increased expression of Rasd2 was alleviated.
- spirulina and phycocyanin were found to have an effect of suppressing muscle atrophy. Moreover, from this result, it was inferred that the spirulina enzymatic hydrolyzate and the phycocyanin enzymatic hydrolyzate also exhibit the effect of inhibiting muscle atrophy.
- compositions containing as an active ingredient at least one selected from the group consisting of spirulina, phycocyanin, spirulina enzymatic degradation products, and phycocyanin enzymatic degradation products increases muscle mass. It was confirmed that the composition exhibited the action of increasing muscle strength, the action of suppressing the decrease in muscle mass, and the action of suppressing muscle atrophy.
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Abstract
Description
また、スピルリナ由来のフィコシアニンが大豆蛋白質よりも高い血清脂質改善作用を有するとして、フィコシアニンを有効成分とする血清脂質低下剤が報告されている(特許文献2参照)。
さらに、スピルリナ(スピルリナ抽出物)を経口投与すると、ナチュラル・キラー(NK)細胞の活性化が誘導されることが報告されている(非特許文献1参照)。
[1]スピルリナ、フィコシアニン、スピルリナ酵素分解物及びフィコシアニン酵素分解物からなる群から選択される少なくとも1種を有効成分として含有する、筋肉量の増加及び低下抑制のための組成物。
[2]上記スピルリナ酵素分解物及び上記フィコシアニン酵素分解物における酵素がプロテアーゼである、[1]に記載の組成物。
[3]スピルリナ、フィコシアニン、スピルリナ酵素分解物及びフィコシアニン酵素分解物からなる群から選択される少なくとも1種を有効成分として含有する、筋肉量の増加及び筋萎縮を抑制するための組成物。
[4]上記スピルリナ酵素分解物及び上記フィコシアニン酵素分解物における酵素がプロテアーゼである、[3]に記載の組成物。
[5]食品組成物である、[1]~[4]のいずれかに記載の組成物。
[6]健康食品、機能性食品、栄養補助食品、サプリメント、保健機能食品、特定保健用食品、栄養機能性食品、機能性表示食品又は病者用食品である、[5]に記載の組成物。
[7]医薬組成物である、[1]~[4]のいずれかに記載の組成物。
[8]サルコペニア又はロコモティブシンドロームの治療、予防又は改善に用いられる医薬組成物である、[7]に記載の組成物。
また、本実施形態に係る組成物は、スピルリナ、フィコシアニン、スピルリナ酵素分解物及びフィコシアニン酵素分解物からなる群から選択される少なくとも1種を有効成分として含有する、筋肉量の増加及び筋萎縮を抑制するための組成物に関する。
以下、本実施形態に係る組成物の有効成分について説明した後、組成物の用途及び形態等について説明する。
スピルリナは、本実施形態に係る組成物における有効成分の一種であり、筋肉量を増加する作用、筋肉量の低下を抑制する作用及び筋萎縮を抑制する作用を有する。
スピルリナは、藍藻類ネンジュモ目ユレモ科スピルリナ属に属する微細なラセン藻であり、豊富なタンパク質、糖類、各種ビタミン、ミネラル、植物性色素を含む。
スピルリナエキスを製造する際に用いる抽出液としては、本発明の効果が得られる範囲で特に限定されるものではないが、例えば、熱水を用いることができる。本実施形態では、藻体のスピルリナを熱水抽出することにより得られる抽出液、或いはその抽出液を濃縮させたり乾燥させて得られる抽出物が好ましく用いることができる。
スピルリナエキスを得る方法としては、特に制限されず、常法に従って得ることができるが、例えば、上記非特許文献1や特開平8-9940号公報等に記載の抽出液の製造方法等を挙げることができる。具体的には、下記に記載の製造方法を挙げることができる。
スピルリナエキスは、スピルリナ藻体を100℃を超える温度で熱水抽出し、当該抽出液のpHを特定の酸性条件下に調整し、その後、不溶性画分を除去することにより、スピルリナ抽出液を得ることで、製造することができる。
スピルリナエキスを液体状態で使用する場合には、上記のようにして得られたスピルリナ抽出液をそのまま使用することができる。また、上記スピルリナ抽出液を濃縮させたり乾燥させ粉末として使用することもできる。本実施形態に係る組成物に用いられるスピルリナエキスとしては、スピルリナ抽出液であっても、スピルリナ抽出液を濃縮或いは乾燥させて製造されたものであってもよい。
スピルリナを培養する際の培養方法としては、藍藻の培養に用いられている通常の方法に従って行うことができる。例えば、屋外において塩基性条件下でスピルリナを培養し、増殖させることができる。
培養して得られたスピルリナ(以下、スピルリナ藻体ともいう)はそのまま用いることもできるし、培養したスピルリナをろ布やろ紙で回収し、水で洗浄後、水に懸濁し懸濁液としてもよい。更に、培養液や懸濁液を濃縮した湿藻体としてもよいし、その湿藻体を凍結乾燥やスプレー乾燥(噴霧乾燥)等により乾燥した乾燥藻体としてもよいし、その乾燥藻体を粉末化してもよい。
熱水抽出に用いるスピルリナ藻体は、湿藻体、凍結乾燥藻体、スプレー乾燥藻体、破砕藻体等いずれでもよい。例えば、破砕藻体を得るには、藻体を通常の方法、例えば工業的にはフレンチ・プレスの様な高圧押し出し法等による破砕処理が挙げられる。
pH調整の際に加える酸としては、硫酸や塩酸でもよいが、現実的な作業工程や食品素材として用いることを考慮すれば、無機酸よりもクエン酸やリンゴ酸等の有機酸を使用することが好ましい。
フィコシアニンは、本実施形態に係る組成物における有効成分の一種であり、筋肉量を増加する作用、筋肉量の低下を抑制する作用及び筋萎縮を抑制する作用を有する。
フィコシアニンは、色素タンパク質であり、発色団としてフィコシアノビリンを有する。フィコシアニンは、フィコシアノビリンとタンパクとが結合した構造を備えている。
また、フィコシアニン調製の原料としては、生の藍藻類を用いてもよく、乾燥処理した藍藻類を用いてもよい。藍藻類の乾燥品としては、生の藍藻類を常法に従い乾燥品としてもよく、市販の乾燥品を用いてもよい。
フィコシアニンを抽出する方法としては、特に制限は無く、常法に従って抽出することができる。
抽出方法の好ましい実施態様としては、例えば、特開2006-230272号公報に記載の抽出方法等を挙げることができる。具体的には、下記抽出方法(i)で記載する抽出方法が挙げられる。下記抽出方法(i)により、高純度であざやかな色調のフィコシアニンを得ることができる。
抽出方法(i)は、
藍藻類中のフィコシアニンを水懸濁液中に抽出させた抽出液を得る第一工程と、
該抽出液中でカルシウム塩とリン酸塩とを反応させてリン酸カルシウムを生成させると共に該リン酸カルシウムにフィコシアニンの夾雑物を吸着させ吸着物を得る第二工程と、
該抽出液から藍藻類の残渣及び吸着物を除去する第三工程と、を有する。
<<フィコシアニンの抽出方法(ii)>>
抽出方法(ii)は、
藍藻類中のフィコシアニンを水懸濁液中に抽出させた抽出液を得る第一工程と、
該抽出液中でカルシウム塩とリン酸塩とを反応させてリン酸カルシウムを生成させると共に該リン酸カルシウムにフィコシアニンの夾雑物を吸着させ吸着物を得る第二工程と、
該抽出液から藍藻類の残渣及び吸着物を除去する第三工程と、
第三工程より前に、抽出液にキレート剤を含有させる工程と、を有する。
特にスピルリナ属の藍藻類に対して、上記抽出方法(i)又は(ii)を用いることにより、C-フィコシアニンとアロフィコシアニンとの混合比が良好な品質のよいフィコシアニンを抽出することができる。
なお、上記抽出方法において、抽出条件を適宜選択することにより、C-フィコシアニンとアロフィコシアニンとの混合比を所望の範囲とするよう調整するとよい。
C-フィコシアニンとアロフィコシアニンとの混合比は、例えば、質量比で3~9.5:0.5~7が好ましく、6~9.5:0.5~4がより好ましく、7~8:2~3がさらに好ましい。
スピルリナ酵素分解物は、本実施形態に係る組成物における有効成分の一種であり、筋肉量を増加する作用、筋肉量の低下を抑制する作用及び筋萎縮を抑制する作用を有する。
スピルリナ酵素分解物は、酵素を作用させ、上述したスピルリナのタンパク質、糖類等を分解させることにより得られる酵素分解物である。
なお、酵素分解に用いる酵素の由来は、特に制限されず、例えば、Aspergillus niger、Aspergillus melleus、Aspergillus oryzae、Rhizopus niveus、Bacillus subtilis、Arthrobacter sp.、Trichoderma viride等を用いることができる。
分子量分布の値は、ゲル濾過カラムを用いて試料を液体クロマトグラフィー分析することにより得ることができる。
スピルリナ水溶液を20~70℃で加熱する。水溶液を用いる酵素の至適pHに調整した後、スピルリナ中のタンパク質に対して、0.01質量%以上、好ましくは0.1~10質量%の酵素を加えて1~24時間攪拌する。攪拌後、加熱や冷却により失活・酵素分解を停止させた後、遠心分離する。遠心分離後、得られた上清を凍結乾燥させてスピルリナ酵素分解物を得る。
フィコシアニン酵素分解物は、本実施形態に係る組成物における有効成分の一種であり、筋肉量を増加する作用、筋肉量の低下を抑制する作用及び筋萎縮を抑制する作用を有する。
フィコシアニン酵素分解物は、酵素を作用させ、上述したフィコシアニンを分解させることにより得られる酵素分解物である。
分子量分布の値は、ゲル濾過カラムを用いて試料を液体クロマトグラフィー分析することにより得ることができる。
フィコシアニン水溶液を20~70℃で加熱する。水溶液を用いる酵素の至適pHに調整した後、フィコシアニンに対して、0.01質量%以上、好ましくは0.1~10質量%の酵素を加えて1~24時間攪拌する。攪拌後、加熱や冷却により失活・酵素分解を停止させた後、遠心分離する。遠心分離後、得られた上清を凍結乾燥させてフィコシアニン酵素分解物を得る。
後述する実施例に示すように、スピルリナ、フィコシアニン、スピルリナ酵素分解物及びフィコシアニン酵素分解物の有効成分は、筋肉量の増加及び低下抑制に関与すると想定される遺伝子の発現抑制を緩和することが認められた。上記有効成分によって発現抑制が緩和される筋肉量の増加及び低下抑制に関与すると想定される遺伝子としては、例えば、Myh13(Myosin, heavy chain 13)、Myh2(Myosin, heavy chain 2)、Myl2(Myosin, light polypeptide 2)、Myh6(Myosin, heavy chain 6)、Tnni1(Troponin I type 1)、Tnnc1((Troponin C type 1)、Itm2a(Integral membrane protein 2A)及びNeu2 (Neuraminidase 2)等が挙げられる。
従って、本実施形態に係る組成物は、スピルリナ、フィコシアニン、スピルリナ酵素分解物及びフィコシアニン酵素分解物からなる群から選択される少なくとも1種を有効成分として含有する、筋肉量の増加及び低下抑制のための組成物として用いることができる。
ここで、ロコモティブシンドロームとは、加齢や運動不足などにより筋肉、骨等の運動器の部位に支障をきたし、日常生活が困難になった状態の症状をいう。ロコモティブシンドロームが運動器全般の症状を含む概念であるのに対し、サルコペニアはその運動器の中でも筋肉量、筋力、身体的機能に着目した症状であるとされている。
後述する実施例に示すように、スピルリナ、フィコシアニン、スピルリナ酵素分解物及びフィコシアニン酵素分解物の有効成分は、筋萎縮に関与すると想定される遺伝子の発現亢進を緩和することが認められた。上記有効成分によって発現が抑制される筋萎縮に関与すると想定される遺伝子としては、例えば、Ddit4(DNA-damage-inducible transcript 4)、Junb(Jun B proto-oncogene)、Egr1(zif-268, Early growth response protein 1、Sdc4(Syndecan 4)、Kcnk5(Potassium channel, subfamily K, member 5)及びRasd2 (Rhes, RASD family, member 2)等が挙げられる。
従って、本実施形態に係る組成物は、スピルリナ、フィコシアニン、スピルリナ酵素分解物及びフィコシアニン酵素分解物からなる群から選択される少なくとも1種を有効成分として含有する、筋肉量の増加及び筋萎縮を抑制するための組成物として用いることができる。
本実施形態に係る組成物は、食品の分野において、一般の食品の他、目的となる作用を有効に発揮できる有効な量の有効成分を食品素材として各種食品に配合することにより、当該作用を有する食品組成物として提供することができる。本実施形態に係る組成物は、例えば、健康食品、機能性食品、栄養補助食品、サプリメント、保健機能食品、特定保健用食品、栄養機能性食品、機能性表示食品、病者用食品、食品添加剤、飼料及び飼料添加剤等の食品組成物に好適に用いることができる。食品組成物の形態としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、固形状、液状、ゲル状等であり得る。
また、食品組成物が、例えば、健康食品、機能性食品、栄養補助食品、サプリメント、保健機能食品、特定保健用食品、栄養機能性食品、機能性表示食品、病者用食品、食品添加剤、飼料及び飼料添加剤等の場合は、錠剤(チュアブル錠等を含む)、カプセル、トローチ、シロップ、ゼリー、顆粒、粉末等であってもよい。
本実施形態に係る組成物は、医薬品の分野において、目的となる作用を有効に発揮できる有効な量の有効成分と共に、薬学的に許容される担体や添加剤等を配合することにより、当該作用を有する医薬組成物として提供することができる。本実施形態に係る組成物は、例えば、上述したサルコペニア又はロコモティブシンドロームの治療、予防又は改善用の医薬組成物に好適に用いることができる。なお、医薬組成物としては、医薬品であっても医薬部外品であってもよい。
医薬組成物の形態としては、特に制限されず、目的に応じて適宜選択することができ、例えば、固形状、液状、ゲル状等であり得る。
食品組成物及び/又は医薬組成物の形態が、例えば、錠剤(タブレットともいう)である場合、有効成分を、賦形剤、結合剤、崩壊剤、滑沢剤、保存剤、酸化防止剤、等張化剤、緩衝剤、コーティング剤、矯味剤、溶解補助剤、基剤、分散剤、安定化剤、着色剤等の添加剤と適宜組み合わせて、常法に従って調製することができる。
緩衝剤としては、炭酸ナトリウム、塩酸、ホウ酸、リン酸塩(リン酸水素ナトリウム等)等が挙げられる。
矯味剤としては、糖類(ブドウ糖、白糖、乳糖等)、サッカリンナトリウム、糖アルコール類等が挙げられる。
安定化剤としては、亜硫酸塩類(亜硫酸水素ナトリウム等)、窒素、二酸化炭素等が挙げられる。
サルコペニアモデルである坐骨神経切除モデルラットを用いて、スピルリナ、フィコシアニン、スピルリナ酵素分解物及びフィコシアニン酵素分解物が、筋肉及び筋萎縮に与える作用を評価した。
被験物質として、スピルリナ、フィコシアニン、スピルリナ酵素分解物及びフィコシアニン酵素分解物を用いた。
屋外の培養池において、塩基性条件下(pH11)でスピルリナ・プラテンシス(Spirulina platensis)を増殖した。次に、増殖したスピルリナ・プラテンシスの噴霧乾燥した藻体粉末50gを、オートクレーブで500mLの蒸留水に懸濁し、圧力を調節することにより、120℃の抽出温度で1時間抽出した。
抽出液をクエン酸にてpHを4.0に調整した。これを、遠心分離にて藻体残渣及びタンパク質(不溶性画分)を除去してスピルリナ熱水抽出液であるスピルリナエキスを得た。
得られたスピルリナエキスを噴霧乾燥後、破砕して、粉末状のスピルリナを得た。
1%塩化カルシウム(無水)溶液1300Lに屋外培養槽で生産したスピルリナ乾燥藻体(噴霧乾燥品)65kgを加え、15分間の攪拌により均一懸濁液とした後、20℃15時間、静置条件下で藍藻類中のフィコシアニンを溶液中に抽出し抽出液を得た。
この抽出液にリン酸二水素ナトリウム32kgを添加し、0.5時間攪拌した後、20℃、静置下で2.5時間反応させ、リン酸カルシウムを生成させると共に該リン酸カルシウムにフィコシアニンの夾雑物を吸着させて吸着物を得た。この後抽出液を遠心分離機に導き、重力加速度が10,000Gで、15分間の遠心分離を行い、藍藻類の残渣及び吸着物を抽出液から除去した。得られたフィコシアニンの抽出液は、分画分子量10,000の分離膜を使用した限外濾過により低分子成分及び塩類を除去した後、トレハロース、クエン酸三ナトリウムを加えて混合し、噴霧乾燥を行い、フィコシアニン色素乾燥物15kgを得た。これを、フィコシアニンとした。なお、フィコシアニンの色素粉末100質量%中、フィコシアニン含量は約30質量%(C-フィコシアニン約22質量%、アロフィコシアニン約8質量%であった。
上記スピルリナ100gを1400mLの蒸留水に溶解させた後、50~52℃に加熱した。次いで、1Nの水酸化ナトリウム溶液を添加し、水溶液のpHを7.0に調整した。スピルリナ中のタンパク質に対して、2質量%のプロチン(SD-NY10、天野エンザイム株式会社製)を水溶液に添加した後、50~52℃で6時間攪拌し、スピルリナを酵素分解させた。反応後の溶液を室温まで冷却した後、遠心分離機に導き、重力加速度が10,000Gで、15分間の遠心分離を行った。上清を採取した後、凍結乾燥させて、スピルリナのプロテアーゼ分解物である、スピルリナ酵素分解物62gを得た。
上記フィコシアニン17gを1400mLの蒸留水に溶解させた後、50~52℃に加熱した。次いで、1Nの塩酸を添加し、水溶液のpHを7.0に調整した。フィコシアニンに対して、2質量%のプロチン(SD-NY10、天野エンザイム株式会社製)を水溶液に添加した後、50~52℃で6時間攪拌し、フィコシアニンを酵素分解させた。反応後の溶液を室温まで冷却した後、遠心分離機に導き、重力加速度が10,000Gで、15分間の遠心分離を行った。上清を採取した後、凍結乾燥させて、フィコシアニンのプロテアーゼ分解物である、フィコシアニン酵素分解物16gを得た。
Slc:SD雄ラット(日本エスエルシー株式会社)を使用した。サルコペニアモデルである坐骨神経切除モデルラットの作製は下記の通りに行った。
被験物質の投与14日目の投与後に、ラットの左後肢を剃毛した後、左大腿部を切開した。筋肉を筋繊維に沿って分割して坐骨神経を露出させた。坐骨神経1cmを切除した後、切開部を縫合し、坐骨神経切除モデルラットを作製した。
実験群、被験物質、投与用量、処置及び例数を下記の表1に示す。投与回数及び投与期間としては、1日1回の頻度で21日間経口投与した(投与液量:10mL/kg)。なお、A1群をSham群(坐骨神経切除を実施していない通常のラット群)、A2群を媒体群ともいう。
A1~A6群において、被験物質の投与21日目、両側後肢の腓腹筋及びヒラメ筋を摘出し、湿重量(g)を測定した。
A1~A6群の各筋の筋肉量(両側後肢の各筋の湿重量(g)/体重(g)、重量%)を算出した。
また、A3~A6群の各筋の平均筋肉量をA2群(媒体群)と比較し、A3~A6群の筋肉量の増加率(%)を算出した(A3~A6群の両側後肢における各筋の平均筋肉量(重量%)/A2群(媒体群)の両側後肢における各筋の平均筋肉量(重量%)×100)。
さらに、A1~A6群の各筋の変化率(左後肢の各筋の平均湿重量(g)/右後肢の各筋の平均湿重量(g)×100、重量%)を算出した。
A1~A3及びA6群それぞれの切除側腓腹筋からRNAを抽出し、DNAマイクロアレイに用いた。DNAマイクロアレイは下記の通りに行った。
筋肉量の増加率の結果を表2に示す。
左後肢(坐骨神経切除)におけるヒラメ筋において、スピルリナ酵素分解物を投与したA4群の筋肉量の増加率が103.7%であり、筋肉量が増加した。
右後肢における腓腹筋において、フィコシアニン酵素分解物を投与したA5群の筋肉量の増加率が102.5%であり、筋肉量が増加した。
右後肢におけるヒラメ筋において、フィコシアニンを投与したA6群の筋肉量の増加率が105.0%であり、筋肉量が増加した。
ここで、被験物質を投与したA3~A6群の変化率の値がA2群の変化率の値と比較して増加していれば、被験物質により筋肉量が増加していると考えられている。
腓腹筋において、スピルリナ酵素分解物を投与したA4群、フィコシアニンを投与したA6の変化率は、A2群に対して増加した。
ヒラメ筋において、スピルリナ酵素分解物を投与したA4群、フィコシアニン酵素分解物を投与したA5群の変化率は、A2群に対して増加した。
<<筋肉量の増加及び低下抑制に関与すると想定される遺伝子>>
筋肉量の増加及び低下抑制に関与すると想定される遺伝子発現量の結果を図1に示す。
図1は、A1群(Sham群)に対する、腓腹筋における筋肉量の増加及び低下抑制に関与すると想定される遺伝子発現量の割合を示す図である。
図1に示すように、腓腹筋においてスピルリナ又はフィコシアニンの投与により、A2群(媒体群)と比較して、筋肉の構造タンパク質である、ミオシン生成に関連するMyh13、Myh2、Myl2、Myh6及びトロポニン生成に関連するTnni1、Tnnc1の発現抑制が緩和していることが認められた。また、筋形成に関する、筋衛星細胞マーカーであるItm2a及び筋形成、筋芽細胞分化に関与するNeu2の発現抑制が緩和していることが認められた。従って、スピルリナ及びフィコシアニンは筋肉量を増加する作用及び筋肉量の低下を抑制する作用を有することが認められた。また、本結果から、スピルリナ酵素分解物及びフィコシアニン酵素分解物においても、同様に筋肉量を増加する作用及び筋肉量の低下を抑制する作用を発揮するものと推察された。
筋萎縮関連遺伝子発現量の結果を図2示す。
図2は、A1群(Sham群)に対する、腓腹筋における筋萎縮に関与すると想定される遺伝子発現量の割合を示す図である。
図2に示すように、腓腹筋においてスピルリナ又はフィコシアニンの投与により、A2群(媒体群)と比較して、筋萎縮及び筋再生に関与するDdit4、Junb、Egr1及び筋再生に関与するSdc4、Kcnk5及びRasd2の発現亢進が緩和していることが認められた。従って、スピルリナ及びフィコシアニンは筋萎縮を抑制する作用を有することが認められた。また、本結果から、スピルリナ酵素分解物及びフィコシアニン酵素分解物においても、同様に筋萎縮を抑制する作用を発揮するものと推察された。
Claims (8)
- スピルリナ、フィコシアニン、スピルリナ酵素分解物及びフィコシアニン酵素分解物からなる群から選択される少なくとも1種を有効成分として含有する、筋肉量の増加及び低下抑制のための組成物。
- 前記スピルリナ酵素分解物及び前記フィコシアニン酵素分解物における酵素がプロテアーゼである、請求項1に記載の組成物。
- スピルリナ、フィコシアニン、スピルリナ酵素分解物及びフィコシアニン酵素分解物からなる群から選択される少なくとも1種を有効成分として含有する、筋肉量の増加及び筋萎縮を抑制するための組成物。
- 前記スピルリナ酵素分解物及び前記フィコシアニン酵素分解物における酵素がプロテアーゼである、請求項3に記載の組成物。
- 食品組成物である、請求項1~4のいずれか1項に記載の組成物。
- 健康食品、機能性食品、栄養補助食品、サプリメント、保健機能食品、特定保健用食品、栄養機能性食品、機能性表示食品又は病者用食品である、請求項5に記載の組成物。
- 医薬組成物である、請求項1~4のいずれか1項に記載の組成物。
- サルコペニア又はロコモティブシンドロームの治療、予防又は改善に用いられる医薬組成物である、請求項7に記載の組成物。
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IN201741006191A (ja) * | 2017-02-21 | 2018-08-24 | Itc Limited | |
KR102076459B1 (ko) * | 2019-10-29 | 2020-02-13 | 주식회사 한미양행 | 갈색거저리 효소처리물을 함유하는 회복기 환자 또는 노약자의 근육량 증강 및 원기회복용 조성물 |
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