Composition for increasing or maintaining muscle mass or muscle strength

Abstract

The present invention provides a novel method capable of improving or maintaining muscle mass or muscle strength, and a novel method capable of preventing obesity, reducing body fat percentage, reducing body fat mass, or reducing fat removal mass. The proteoglycan applied in the invention is obtained by the preparation method of the proteoglycan, and the preparation method comprises the following steps: dipping a biological sample containing the proteoglycan in an aqueous solution of a surfactant, and recovering the dipped solution.

Description

Technical Field

[0001] The present invention relates to a composition and method containing specific proteoglycans for increasing or maintaining muscle mass or muscle strength, as well as a composition and method for preventing obesity, reducing body fat percentage, reducing body fat mass or reducing fat-free mass. Background Art

[0002] In Japan, there is a difference of about 10 years between the average life expectancy and the healthy life expectancy, and the extension of the healthy life expectancy is a pressing issue. Among the main reasons why nursing care is necessary, the reduction in the function of the motor organs due to joint diseases, weakness caused by advanced age, fractures / falls, etc., accounts for about 35% of the total, and if limited to those who need support, it accounts for about 50%. In other words, it is believed that in order to extend the healthy life expectancy, it is important to keep the motor organs (skeletal muscles, joints, bones) healthy. However, it is well known that muscle mass decreases with age, and it is known that in addition to aging, the reduction in activities of daily living (ADL) accompanied by inflammation, incoordination and pain in the knees also leads to a decrease in muscle mass and muscle strength.

[0003] Proteoglycan is a general term for glycoproteins having extremely complex and diverse structures in which several to dozens of linear sugar chains are covalently bonded to one core protein. Chondroitin sulfate is a representative sugar chain contained in proteoglycans present in cartilage tissue.

[0004] Chondroitin sulfate is an ingredient that has attracted industry attention due to its high usefulness, such as excellent moisturizing properties, biocompatibility, and lubricity, and various methods for efficiently recovering and preparing it from natural resources have been developed.

[0005] In cartilage tissue, chondroitin sulfate itself does not exist alone, but exists in the form of a complex formed with protein via a covalent bond, namely proteoglycan. However, it is often difficult to extract proteoglycan as it is due to the complex structure of the glycoprotein complex, so the method of completely decomposing the core protein part of proteoglycan and extracting only chondroitin sulfate is mainly adopted. The prepared product of this method is mucopolysaccharides such as chondroitin sulfate.

[0006] On the other hand, attempts have also been made to recover and prepare proteoglycans as they are, rather than as chondroitin sulfate, and to utilize them. In particular, the cartilage tissues of fish, birds, and mammals contain proteoglycans with chondroitin sulfate as the main sugar chain. On the other hand, since these cartilage tissues are usually disposed of, several methods for preparing proteoglycans from cartilage tissues that take into account the effective utilization of waste have been proposed.

[0007] For example, as a method for preparing proteoglycan from cartilage tissue, Patent Document 1 describes a method for preparing proteoglycan, which includes immersing a biological sample such as cartilage tissue containing proteoglycan in an aqueous solution of a surfactant and recovering the immersion solution. In addition, it is known that the proteoglycan described in Patent Document 1 is useful in preventing, alleviating or treating the symptoms of arthritis (Patent Document 2).

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: Japanese Patent Application Publication No. 2014-9164

[0011] Patent Document 2: Japanese Patent Application Publication No. 2018-58781 Summary of the invention

[0012] Problem that the invention aims to solve

[0013] As described above, extension of healthy life span is a pressing issue, and in order to extend healthy life span, it is considered useful to prevent a decrease in muscle mass or muscle strength, that is, to increase or maintain muscle mass or muscle strength.

[0014] Means used to solve problems

[0015] The present inventors conducted intensive research in view of the above problems and found that proteoglycans prepared by a specific method can increase or maintain muscle mass or muscle strength and can prevent obesity, reduce body fat percentage, reduce body fat mass or reduce fat-free mass, thereby completing the present invention.

[0016] Therefore, the present invention provides the following solutions.

[0017] [1] A composition for increasing or maintaining muscle mass or strength, the composition containing proteoglycan, wherein the proteoglycan is obtained by a method for preparing proteoglycan, the method comprising: immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant, and recovering the immersion solution.

[0018] [2] A composition for preventing obesity, reducing body fat percentage, reducing body fat mass, or reducing fat-free mass, the composition containing proteoglycan, the proteoglycan being obtained by a proteoglycan preparation method, the preparation method comprising: a step of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant, and a step of recovering the immersion solution.

[0019] [3] The composition according to [1] or [2], wherein the surfactant is at least one selected from the group consisting of saponin, lecithin, and sucrose fatty acid esters.

[0020] [4] The composition according to any one of [1] to [3], wherein the biological sample containing proteoglycan is at least one selected from the group consisting of cartilage tissue, muscle fiber and skin of fish, mollusk, bird and mammal.

[0021] [5] The composition according to any one of [1] to [4], wherein the biological sample containing proteoglycan is at least one selected from the group consisting of cartilage tissues of fish, birds, and mammals.

[0022] [6] The composition according to any one of [1] to [5], which is used for increasing or maintaining muscle mass or muscle strength in healthy humans.

[0023] [7] The composition according to any one of [1] to [6], which is a cosmetic composition, a food composition, or a pharmaceutical composition.

[0024] [8] A method for increasing or maintaining muscle mass or muscle strength, the method comprising administering an effective amount of proteoglycan to a subject in need of proteoglycan, the proteoglycan being obtained by a proteoglycan preparation method, the preparation method comprising: a step of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant, and a step of recovering the immersion solution.

[0025] [9] A method for preventing obesity, reducing body fat percentage, reducing body fat mass, or reducing fat-free mass, the method comprising administering an effective amount of proteoglycan to a subject in need of proteoglycan, the proteoglycan being obtained by a proteoglycan preparation method, the preparation method comprising: a step of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant, and a step of recovering the immersion solution.

[0026]

[10] The method according to [8] or [9], wherein the surfactant is at least one selected from the group consisting of saponin, lecithin, and sucrose fatty acid esters.

[0027]

[11] The method described in [8] or [9], wherein the biological sample containing proteoglycan is at least one selected from the group consisting of cartilage tissue, muscle fiber and skin of fish, mollusk, bird and mammal.

[0028]

[12] The method described in

[11] , wherein the biological sample containing proteoglycan is at least one selected from the group consisting of cartilage tissue of fish, birds and mammals.

[0029]

[13] The method described in [8] is used to increase or maintain muscle mass or muscle strength in healthy subjects.

[0030]

[14] A proteoglycan for use in increasing or maintaining muscle mass or muscle strength, wherein the proteoglycan is obtained by a proteoglycan preparation method, the preparation method comprising: immersing a biological sample containing the proteoglycan in an aqueous solution of a surfactant, and recovering the immersion solution.

[0031]

[15] A proteoglycan for use in preventing obesity, reducing body fat percentage, reducing body fat mass, or reducing fat-free mass, wherein the proteoglycan is obtained by a proteoglycan preparation method, the preparation method comprising: a step of immersing a biological sample containing the proteoglycan in an aqueous solution of a surfactant, and a step of recovering the immersion solution.

[0032]

[16] The proteoglycan according to

[14] or

[15] , wherein the surfactant is at least one selected from the group consisting of saponin, lecithin, and sucrose fatty acid esters.

[0033]

[17] The proteoglycan described in

[14] or

[15] , wherein the biological sample containing the proteoglycan is at least one selected from the group consisting of cartilage tissue, muscle fiber and skin of fish, molluscs, birds and mammals.

[0034]

[18] The proteoglycan described in

[17] , wherein the biological sample containing the proteoglycan is at least one selected from the group consisting of cartilage tissues of fish, birds and mammals.

[0035]

[19] The proteoglycan described in

[14] is used for increasing or maintaining muscle mass or muscle strength in healthy humans.

[0036]

[20] A method for preparing a proteoglycan for increasing or maintaining muscle mass or strength, wherein the proteoglycan is obtained by a method for preparing the proteoglycan, the method comprising: immersing a biological sample containing the proteoglycan in an aqueous solution of a surfactant, and recovering the immersion solution.

[0037]

[21] Application of proteoglycan for preventing obesity, reducing body fat percentage, reducing body fat mass, or reducing fat-free mass, wherein the proteoglycan is obtained by a proteoglycan preparation method, the preparation method comprising: a step of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant, and a step of recovering the immersion solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] [ Figure 1 ] Figure 1 Test results showing muscle mass.

[0039] [ Figure 2 ] Figure 2The test results of body measurement excluding muscle mass, grip strength, 10m walking test, Time up and go Test, and blood test (baseline) are shown.

[0040] [ Figure 3 ] Figure 3 The test results of body measurement excluding muscle mass, grip strength, 10m walking test, timed stand-and-go test, and blood test (actual measured values) are displayed.

[0041] [ Figure 4 ] Figure 4 The test results of body measurement excluding muscle mass, grip strength, 10m walk test, timed stand-and-go test, and blood test (variation) are displayed. DETAILED DESCRIPTION

[0042] The proteoglycan used in the present invention (hereinafter also referred to as "the proteoglycan of the present invention") is a glycoprotein having a very complex and diverse structure. Since it is impossible or completely impractical to express its structure by a structural formula, etc., in the present invention, the proteoglycan is specified by the production method.

[0043] The proteoglycan of the present invention can be produced based on the method described in JP-A-2014-9164, and the method is described below.

[0044] The method for producing the proteoglycan of the present invention comprises the steps of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant and recovering the solution after the immersion.

[0045] As biological samples containing proteoglycans, cartilage tissues, muscle fibers and skins of fish, molluscs, birds and mammals are listed, and cartilage tissues are particularly preferred. As cartilage tissues, cartilage tissues of fish, birds and mammals, and any of the discarded parts of the cartilage tissues can be used. Here, cartilage tissues include cartilage alone or any of the tissues of the peripheral parts containing cartilage, such as bones, muscle fibers, skin, etc. As cartilage tissues of fish, it is needless to say that the fins and cartilage parts of cartilaginous fish such as blue sharks and mako sharks that are commonly circulated are particularly preferred. The nasal cartilage tissues called ice heads, which contain about 6% of their average weight in the head of salmon, are preferably used. When salmon (mostly whitefish) caught on the coast of Hokkaido are processed as various processed products, their heads are usually not needed, so the cut heads are partially processed into fish meal and used, but most of them are discarded as industrial waste, and ice heads can be obtained from such wastes simply, cheaply and stably.

[0046] In the present invention, in addition to ice head, cartilage tissues of fish sources such as the cartilage tissue of skates and sharks, cartilage tissues of birds such as chickens, and cartilage tissues of mammals such as laryngeal cartilage and bronchial cartilage of cattle and cartilage of whales can also be used. Further, it is known that proteoglycans are also present in the epidermis / cartilage tissues of squid and octopus as molluscs, and the epidermis / cartilage of these molluscs can also be applied in the present invention. In addition, it is reported that there is a chondroitin / protein complex (Bikunin) that is almost free of sulfuric acid in mammalian blood, and this chondroitin accounts for almost 100% of the mucopolysaccharides in mammalian blood (Salier JP, Rouet P., Raguenez G., Daveau M.: The inter-alpha-inhibitor family: from structure to regulation Biochem. J., 315, 1-9, 1996), so the chondroitin of mammalian blood source can also be used as a biological sample containing proteoglycans in the present invention. Many of the above-mentioned biological samples containing proteoglycans are industrial wastes and are easy to obtain. Before being immersed in the surfactant aqueous solution, these raw materials are preferably subjected to pretreatment such as crushing as finely as possible and defatting in order to increase the surface area and improve the extraction amount of proteoglycans.

[0047] As the surfactant, any surfactant such as anionic surfactant, cationic surfactant, amphoteric surfactant and nonionic surfactant can be used. Glycerol fatty acid ester, polyglycerol fatty acid ester, propylene glycol fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, soybean lecithin (lecithin), saponin etc. which are approved as food additives or food emulsifiers are particularly preferred, and saponin, lecithin or sucrose fatty acid ester monomers or any mixture thereof are particularly preferred.

[0048] Examples of saponin include quillaja saponin, soybean saponin, and tea saponin, and quillaja saponin is particularly preferred.

[0049] Examples of lecithin include vegetable lecithin, fractionated lecithin, egg yolk lecithin, enzyme-treated lecithin, and enzymatically hydrolyzed lecithin. Vegetable lecithin having an HLB of 3 to 4 is particularly preferred.

[0050] Sucrose fatty acid esters include sucrose fatty acid monoesters in which one molecule of fatty acid can be added to one molecule of sucrose, diesters in which two molecules of fatty acid can be added, and triesters in which three molecules of fatty acid can be added. Theoretically, as sucrose fatty acid esters, there can be octaesters in which up to eight molecules of fatty acid can be added, but suitable for food use are monoesters, diesters, triesters, and mixtures of any of them. From the viewpoint of emulsification stability of the emulsion, sucrose fatty acid esters with HLB 13 to 19, particularly 14 to 19, and optimally HLB 19 are preferred. If the viewpoint of reducing the cost of the emulsion is added, a mixture of monoesters, diesters, and triesters (hereinafter also referred to as "sucrose fatty acid mixed esters") is desired, and in particular, a mixture of sucrose fatty acid mixed esters with HLB 14 to 16 and sucrose fatty acid monoesters with HLB 19 are desired. As fatty acids, saturated or unsaturated fatty acids with 10 to 22 carbon atoms are preferred, and saturated or unsaturated fatty acids with 12 to 18 carbon atoms are more preferred.

[0051] Among the surfactants used in the present invention, sucrose fatty acid esters are preferred in view of the extraction efficiency of proteoglycans and the simplicity of post-treatment. In particular, sucrose fatty acid ester of lauric acid, which is a saturated fatty acid having 12 carbon atoms, i.e., sucrose laurate is preferred.

[0052] The concentration of the surfactant in the surfactant aqueous solution is preferably 0.01 to 20 weight %, particularly preferably 0.1 to 10 weight %, and further preferably 0.5 to 5 weight %. The immersion time can be appropriately set as needed, and the immersion time in the case of an aqueous solution of, for example, 0.1 to 2 weight % of a surfactant, for example saponin, lecithin or sucrose fatty acid ester, or any mixture thereof is preferably more than 10 hours. In addition, the immersion time in the case of a solution of 2 to 5 weight % of a surfactant, for example saponin, lecithin or sucrose fatty acid ester, or any mixture thereof is preferably about 12 hours. By these conditions, it is possible to effectively reclaim and prepare proteoglycans of higher molecular weight.

[0053] The immersion of the biological sample in the aqueous surfactant solution is preferably performed at 30° C. to 100° C., more preferably 40° C. to 80° C., and particularly preferably 50° C. to 70° C. In particular, by setting the immersion temperature to 50° C. to 60° C., proteoglycan is hardly decomposed, and can be extracted as a high-molecular glycoprotein complex.

[0054] The immersion is preferably performed with 1 to 50 parts by weight, more preferably 2 to 30 parts by weight, further preferably 4 to 20 parts by weight, and particularly preferably 6 to 15 parts by weight of an aqueous surfactant solution relative to 1 part by weight of cartilage. The immersion is preferably performed while stirring with a mixer or a stirrer.

[0055] The extraction of proteoglycan from cartilage tissue can be monitored by detecting or quantifying the amount of uronic acid, for example, by the carbazole method (John et al., ANALYTICALBIOCHEMISTRY, 1967, Vol. 19, pp. 119-132), or by detecting and monitoring uronic acid by other known methods.

[0056] Since the surfactant aqueous solution after immersion contains a large amount of residues after proteoglycan extraction, it is preferable to remove these by other methods such as filtration and centrifugation.

[0057] The extract containing proteoglycan may be used as a product as it is, but it is preferred to separate or purify the proteoglycan by an appropriate method until the purity required for various uses of the proteoglycan is achieved.

[0058] In the present invention, the purification method of proteoglycan can be applied to a known purification method. As a preferred purification method, centrifugation can be cited. By centrifugation, fine solids can be easily removed as precipitated residues.

[0059] In addition, the liquid phase containing proteoglycan recovered by centrifugation can also be further filtered by using filter paper or an ultrafiltration membrane separation device with an appropriate fractional molecular weight. As an exclusion molecular weight, a range of about 50,000 to 1,000,000 is sufficient. In this operation, if a fractional molecular weight of more than 500,000 is used, collagen can also be removed from the liquid phase, which can improve the purity of proteoglycan. In addition, by adding water to the liquid phase containing proteoglycan and reducing the viscosity of the liquid phase, while making the passage of the membrane easier, by repeating it, a specific smell of the raw material source generated in small quantities can also be removed.

[0060] Furthermore, the obtained concentrated solution can be added to salt-saturated ethanol to recover gel-like proteoglycan, which can be made into a solid by using a vacuum freeze dryer or dried by a spray dryer to form a powdered solid.

[0061] As long as the purpose of the present invention can be achieved, the molecular weight of the proteoglycan of the present invention is not particularly limited, and is preferably, for example, 1 million or more, preferably 1.5 million or more, and more preferably 1.8 million or more. The upper limit is not particularly limited, and is preferably, for example, about 3 million or less.

[0062] As a method for measuring the molecular weight, a method using a high performance liquid chromatography apparatus is mentioned.

[0063] The composition containing the proteoglycan of the present invention (hereinafter also referred to as the composition of the present invention) can increase or maintain muscle mass or muscle strength by applying it to a subject.

[0064] "Increasing or maintaining muscle mass or muscle strength" means increasing or maintaining the muscle mass or muscle strength of the subject compared to the state before application. Therefore, the composition of the present invention can also prevent a decrease in muscle mass or a decrease in muscle strength.

[0065] The method for measuring muscle mass or muscle strength is not particularly limited, and for example, an instrument or device that can measure the muscle mass or muscle strength of a subject can be used for measurement. As such an instrument or device, for example, in the case of measuring the muscle strength of an arm, a commercially available handgrip dynamometer can be used, and in the case of measuring the muscle mass of the whole body, a commercially available body composition meter can be used.

[0066] The target muscles are not particularly limited, and muscles throughout the body may be targeted.

[0067] For example, in the case of facial muscles, it is possible to prevent a decrease in facial muscle mass or strength, and as a result, wrinkles or sagging can be prevented.

[0068] For example, in the case of skeletal muscles, since it is possible to prevent a decrease in muscle mass or strength of the muscles that make the body move, bedriddenness and movement disorder syndrome can be prevented. It is also related to preventing obesity, reducing body fat percentage, reducing body fat mass, or reducing fat-free mass.

[0069] The application object of the composition of the present invention is not particularly limited, and examples thereof include mammals such as mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, cattle, horses, sheep, monkeys, and humans. The application object of the composition of the present invention is preferably a human being, more preferably a healthy human being, and particularly preferably a healthy human being who has felt a decrease in muscle mass or muscle strength due to aging.

[0070] The method of applying the composition of the present invention to a subject is not particularly limited, and the composition can be applied orally or parenterally. Therefore, the composition of the present invention can be prepared into, for example, a cosmetic composition, a food composition, and a pharmaceutical composition.

[0071] The composition of the present invention may contain inert ingredients known or well-known in the preparation of cosmetic compositions, food compositions, and pharmaceutical compositions as required, and may also contain active ingredients other than the proteoglycan of the present invention in order to enhance or otherwise enhance the effects.

[0072] The inert ingredients that may be included in the composition of the present invention are not particularly limited as long as the effects of the present invention are achieved, and examples thereof include carriers permitted as cosmetics, foods or drugs, such as excipients, binders, extenders, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, film-forming agents, carriers, diluents, and the like.

[0073] The specific application amount and application interval of the composition of the present invention can vary according to the weight, sex, age, state, application route or other factors of the object. The specific application amount and application interval can be appropriately determined by those skilled in the art. However, if non-limiting specific examples are listed, usually, in the case of oral administration, relative to 1 adult (60kg), the daily intake of the proteoglycan of the present invention is, for example, more than 5 mg, preferably more than 10 mg, more preferably more than 20 mg, further preferably more than 30 mg, preferably less than 1000 mg, more preferably less than 500 mg, further preferably 200 mg, further preferably less than 100 mg, further more preferably less than 80 mg, and particularly preferably 40 mg. In the present invention, it is preferred that such an amount be applied once a day to multiple times.

[0074] The application period is not particularly limited, but repeated or continuous application is preferred, continuous application for 5 days or more is more preferred, continuous application for 12 weeks or more is further preferred, and continuous application for 24 weeks or more is particularly preferred.

[0075] There is no particular limitation on the specific form of the composition of the present invention as long as the effects of the present invention can be achieved.

[0076] When the composition of the present invention is used as a cosmetic composition or a non-oral pharmaceutical composition, for example, it can be used in the form of a skin external preparation. Examples of skin external preparations include creams, lotions, emulsions, foundations, masks, foams, plasters, ointments, papules, aerosols, and the like. When used as a non-oral pharmaceutical composition, for example, it can also be used in the form of an injection, and the like.

[0077] When the composition of the present invention is used as a food composition or an oral pharmaceutical composition, for example, it can be used in the form of tablets, capsules, granules, powders, lozenges, liquids, syrups, suspensions, etc. In addition, the form of the food composition also includes refreshing drinks, tea drinks, coffee drinks, fruit juice drinks, carbonated drinks, milk drinks, jellies, wafers, biscuits, bread, noodles, sausages, etc.

[0078] As the form of the food composition or the pharmaceutical composition, solid preparations such as tablets, capsules, granules, powders, and lozenges are preferred. Since they can be easily ingested, tablets and capsules are more preferred, and tablets are particularly preferred.

[0079] In the case where the composition of the present invention is formulated into tablets, it is preferred to contain crystalline cellulose and a lubricant.

[0080] The content of crystalline cellulose in the tablet is 1 to 80% by weight, preferably 3 to 75% by weight. If the content of crystalline cellulose in the tablet is too high, sticking is likely to occur. On the contrary, if the content of crystalline cellulose is too low, the hardness of the tablet decreases.

[0081] As the lubricant contained in the tablet, commonly used lubricants such as calcium stearate, silicon dioxide, carboxymethyl cellulose calcium, sucrose fatty acid ester, calcium silicate, magnesium silicate, talc and silica hydrogel are listed, and calcium stearate, silicon dioxide and carboxymethyl cellulose calcium are particularly preferred. The content of the lubricant in the tablet is 0.5-5% by mass, preferably 0.5-3% by mass. If the lubricant content in the tablet is too small, obstacles such as adhesion and capping are likely to occur during the preparation of the tablet. On the contrary, if the lubricant content is too much, the tablet's formability deteriorates and sufficient tablet hardness cannot be obtained.

[0082] Tablets can be prepared by uniformly mixing and compressing the raw materials simultaneously or sequentially. Compression molding generally refers to a preparation method by tableting, including a granule tableting method in which granules are made from a raw material mixed powder and then compressed and formed, and a direct tableting method in which the raw material mixed powder is directly compressed and formed as it is. Since the tablet of the present invention has good tableting properties, any of the granule tableting method and the direct tableting method can be applied as its preparation method. From the perspective of simple steps, the direct tableting method is preferred. Tableting can be performed using a continuous tableting machine such as a single-shot tablet press and a rotary tablet press according to conventional methods. The tableting pressure can be appropriately set in consideration of the size and hardness of the desired tablet described later, for example, it can be set to 2000 to 4000 kgf.

[0083] The size of the tablet can be any general size, for example, a diameter of about 5 to 12 mm and a mass of about 100 to 400 mg. Considering the ease of taking, the diameter is preferably about 5 to 10 mm and the mass is about 200 to 300 mg. The hardness of the tablet can be any general tablet hardness, for example, more than 6 kgf. Considering the suppression of damage such as cracks and notches, the smoothness of the surface, the disintegration after ingestion, etc., it is preferably about 6.5 to 15 kgf. The hardness of the tablet can be measured using a general tablet hardness meter (for example, Freund's tablet hardness meter).

[0084] The present invention is further described in detail with reference to the following preparation examples and embodiments, but the present invention is not limited to these embodiments.

[0085] Example

[0086] [Preparation Example 1]

[0087] The nasal cartilage removed from the head of a whitefish stored frozen at -40°C is crushed into fine pieces using an electric meat slicer. The fine pieces of nasal cartilage are defatted and dehydrated. The treated nasal cartilage is dried by ventilation or reduced pressure, and 24 g is used as the starting material. 2970 g of distilled water pre-cooled to 5°C is placed in a 5-liter extraction container, and 30 g of the solid sucrose fatty acid ester used in Preparation Example 1 is further added to prepare a total of 3000 g (1 weight %) of sucrose fatty acid ester aqueous solution. 24 g of the starting material is added to the extraction container, and the mixture is immersed for 12 hours while being stirred with a stirrer.

[0088] After the immersion was completed, the contents were transferred to another container equipped with a 1 mm square stainless steel filter, the nasal cartilage was removed, and the extract containing proteoglycan was recovered.

[0089] The extract was centrifuged at 3000 rpm for 20 minutes using a Hitachi himac CF7D2 centrifuge to remove the solid and oil fractions and recover the liquid phase containing proteoglycan.

[0090] Furthermore, the liquid phase was filtered using filter paper (manufactured by Advantec), and after adding distilled water 6 times the amount of the filtrate, fractionation and concentration were simultaneously performed using BIOMAX 100K POLYETHERSULFONE (cutoff molecular weight 100,000) manufactured by Japan Millipore.

[0091] A portion of the obtained concentrated liquid was collected and the weight of the solid content in the liquid was measured. For the measurement, the solid content was precisely measured using a digital meter (GF-400 of A&D Co.) after drying at 105°C for 16 hours in a drying oven (YAMATO DX401) to completely evaporate the water. As a result, 5.5 g of dry solid content, which was equivalent to 22.21% of the starting material in terms of conversion value, was obtained from 24 g of the starting material.

[0092] In addition, by using an amino acid automatic analyzer (Hitachi, Ltd., L-8500 Amino Acid Analyzer) to measure the amino acid amount in the concentrate and quantitatively measure the collagen amount in the concentrate, the proteoglycan amount is calculated by quantitatively measuring the uronic acid amount by the Galambos method. Further, a high performance liquid chromatography device (Shimadzu, column TSK-GELG4000PWXL) is used to measure the molecular weight of proteoglycan.

[0093] The results of these analyses showed that the solid content contained 14.8% protein, 18.4% ash, 64.8% carbohydrates, and 0% lipids. In addition, the molecular weight of proteoglycan was about 1.3 million.

[0094] [Preparation Example 2]

[0095] The nasal cartilage removed from the head of a whitefish stored frozen at -40°C is crushed into fine pieces using an electric meat cutter. The fine pieces of nasal cartilage are defatted and dehydrated. The treated nasal cartilage is dried by ventilation or reduced pressure, and 17.9 g is used as the starting material. 2495 g of distilled water pre-cooled to 5°C is placed in a 5-liter extraction container, and 5 g of solid Quillaja saponin is further added to prepare a total of 2500 g (0.2 weight %) of saponin aqueous solution. 17.9 g of the starting material is added to the extraction container, and the mixture is immersed for 12 hours while being stirred with a stirrer.

[0096] After the immersion was completed, the contents were transferred to another container equipped with a 1 mm square stainless steel filter, the nasal cartilage was removed, and the extract containing proteoglycan was recovered.

[0097] The extract was centrifuged at 3000 rpm for 20 minutes using a Hitachi himac CF7D2 centrifuge to remove the solid and oil fractions and recover the liquid phase containing proteoglycan.

[0098] Furthermore, the liquid phase was filtered using filter paper (manufactured by Advantec), and after adding distilled water 6 times the amount of the filtrate, fractionation and concentration were simultaneously performed using BIOMAX 100K POLYETHERSULFONE (cutoff molecular weight 100,000) manufactured by Japan Millipore.

[0099] A portion of the obtained concentrated liquid was collected and the weight of the solid content in the liquid was measured. For the measurement, the solid content was precisely measured using a digital meter (GF-400 of A&D Co.) after drying at 105°C for 16 hours in a drying oven (YAMATO DX401) to completely evaporate the water. As a result, 3.29 g of dry solid content, which was equivalent to 18.41% of the starting material in terms of conversion value, was obtained from 17.9 g of the starting material.

[0100] In addition, by using an amino acid automatic analyzer (Hitachi, Ltd., L-8500 Amino Acid Analyzer) to measure the amino acid amount in the concentrate and quantitatively measure the collagen amount in the concentrate, the proteoglycan amount is calculated by quantitatively measuring the uronic acid amount by the Galambos method. Further, a high performance liquid chromatography device (Shimadzu, column TSK-GELG4000PWXL) is used to measure the molecular weight of proteoglycan.

[0101] The results of these analyses showed that the solid content contained 17% protein, 22.4% ash, 60.6% carbohydrates, and 0% lipids. In addition, the molecular weight of proteoglycan was about 1.2 million.

[0102] [Preparation Example 3]

[0103] The cartilage removed from the blue mackerel stored frozen at -40°C was crushed into a fine powder using an electric meat cutter, and 200 g of the powder was prepared as the starting material. 2376 g of distilled water pre-cooled to 0°C was placed in a 5-liter extraction container, and 24 g of solid sucrose fatty acid ester (C12; HLB14-18) was further added to prepare a total of 2400 g (1% by weight) of sucrose fatty acid ester aqueous solution. 200 g of the starting material was placed in the extraction container, and the mixture was immersed for 12 hours while being stirred with a stirrer.

[0104] After the immersion was completed, the contents were transferred to another container equipped with a 1 mm square stainless steel filter, and the extract containing proteoglycan was recovered.

[0105] The extract was centrifuged at 1500×g for 30 minutes using an IWAKI CFS-400 centrifuge to remove the solid and oil fractions and recover the proteoglycan-containing liquid phase.

[0106] The liquid phase was further filtered using filter paper (manufactured by Advantec), and after adding distilled water 6 times the amount of the filtrate, it was simultaneously fractionated and concentrated using a PREP / SCALE TFF membrane (molecular weight cutoff 100,000) manufactured by Millipore Japan.

[0107] A portion of the obtained concentrated liquid was collected and the weight of the solid content in the liquid was measured. For the measurement, the solid content was precisely measured using a digital meter (GF-400 of A&D Co.) after the water was completely evaporated at 105°C for 16 hours by drying in a drying oven (YAMATO DX401). As a result, 5.5 g of dry solid content, which was equivalent to 2.75% of the starting material in terms of conversion value, was obtained from 200 g of the starting material.

[0108] The amount of collagen in the concentrate was quantified by measuring the amount of amino acids in the concentrate using an automatic amino acid analyzer (L-8500 Amino Acid Analyzer, manufactured by Hitachi, Ltd.), and the amount of uronic acid was quantified by the carbazole method to calculate the amount of proteoglycan.

[0109] Furthermore, the molecular weight of proteoglycan was measured using a high performance liquid chromatography apparatus (Shimadzu Corporation, column Asahipak).

[0110] The results of these analyses determined that there were 35% protein, 21.5% ash, 42.9% carbohydrates, and 0.6% lipids in the solid portion. According to Patent Document 1, the weight ratio of the core protein of proteoglycan is recorded as about 7%, so the proteoglycan in the present invention is calculated to be about 42.9% based on carbohydrates, and the purity is estimated to be about 46%. In addition, the molecular weight of proteoglycan is about 2 million.

[0111] In the operation shown in the above-mentioned Preparation Example 3, the concentration of the sucrose fatty acid ester solution was changed to 0.1%, 5%, and 10%, and immersion and extraction were performed for 24 hours. The results of studying the time-dependent changes in the amount of proteoglycan recovered (uronic acid amount) at this time obtained roughly the same results.

[0112] In addition, in the operation shown in the above-mentioned Preparation Example 1, sucrose fatty acid ester was changed to 0.666N acetic acid, and immersion and extraction were performed for 24 hours. The time-dependent changes in the recovery amount of proteoglycan (uronic acid amount) at this time were studied, and the results were worse than those of Preparation Example 3.

[0113] [Example]

[0114] The effect of the composition of the present invention was tested by a placebo-controlled randomized double-blind parallel group comparison method.

[0115] 1. Formulation of the test composition

[0116] In the following examples, the proteoglycan prepared in Preparation Example 1 (hereinafter, also referred to as "proteoglycan complex") and the test composition having the following formulation were used.

[0117] [Table 1]

[0118]

[0119] The raw materials are mixed in such a ratio that one tablet has the above composition, and the obtained mixture is directly compressed to prepare tablets.

[0120] 2. Trial period

[0121] 24 weeks

[0122] 3. Application method

[0123] The subjects were asked to take 1 tablet once a day with water or hot water after dinner.

[0124] 4. Subjects

[0125] The following persons were selected as subjects.

[0126] (1) Registration criteria

[0127] i. Japanese

[0128] ii. Adult men and women

[0129] iii. Healthy people

[0130] iv. People who feel weak

[0131] (2) Exclusion criteria

[0132] i. Patients who are currently being treated for or have a history of malignant tumors, heart failure, or myocardial infarction

[0133] ii. Patients with pacemakers or implantable defibrillators

[0134] iii. Those who are undergoing treatment for the following chronic diseases

[0135] Arrhythmia, liver damage, kidney damage, cerebrovascular damage, rheumatism, diabetes,

[0136] Dyslipidemia, hypertension, other chronic diseases

[0137] iv. Those diagnosed with sarcopenia

[0138] v. People who usually take specific health foods, functional label foods, and other foods / beverages that are considered functional

[0139] vi. Commonly used medicines (including Chinese medicine) / supplements

[0140] vii. Those who have allergies (drugs / test food related foods)

[0141] viii. Those who are pregnant, breastfeeding, or intend to become pregnant during the trial period

[0142] ix. Those who have participated in other clinical trials within 3 months before the date of obtaining consent, or those who plan to participate during the trial

[0143] x. Other persons who are judged by the responsible physician to be unsuitable as subjects of this trial

[0144] (3) Selection criteria

[0145] i. Those who are judged by the responsible physician to have no problem participating in the trial

[0146] ii. Excluded if two or more of the following criteria are met.

[0147] a. Grip strength is less than 28kg for men and less than 18kg for women

[0148] b. Those whose normal walking speed is less than 1m / s

[0149] c. Skeletal muscle mass is less than 7.0 kg / m for men 2 , women less than 5.7kg / m 2 Those

[0150] iii. Those with relatively low muscle mass

[0151] 5. Evaluation Project

[0152] Rate the following items.

[0153] (1) Grip strength

[0154] i. Implementation content: Using handgrip dynamometer, study handgrip strength in implementing medical institutions.

[0155] ii. Research Project: Grip Strength

[0156] iii. Evaluation method: Screening and examination before intake, examination after 12 weeks of intake and examination after 24 weeks of intake were used for measurement.

[0157] (2) 10m walking test

[0158] i. Implementation content: Conduct a 10-meter walking test to evaluate walking ability.

[0159] ii. Research items: walking time, number of steps, stride length, walking rate (number of steps per unit time)

[0160] iii. Evaluation method: Screening and examination before intake, examination after 12 weeks of intake and examination after 24 weeks of intake were used for measurement.

[0161] (3) Time up and go Test

[0162] i. Implementation content: Conduct a timed stand-and-go test to evaluate walking ability.

[0163] ii. Research items: normal walking speed, maximum walking speed

[0164] iii. Evaluation method: Screening and examination before intake, examination after 12 weeks of intake and examination after 24 weeks of intake were used for measurement.

[0165] 6. Results

[0166] (1) Effectiveness evaluation items

[0167] i. Main results

[0168] a. Actual measured value of muscle mass after 24 weeks of intake

[0169] ii. Secondary outcomes

[0170] a. Actual measured value of muscle mass after 12 weeks of intake

[0171] b. Changes in muscle mass after 12 weeks and 24 weeks of intake from the screening and pre-intake examination

[0172] c. Grip strength, walking time, number of steps, stride length, walking rate, normal walking speed, maximum walking speed, body fat percentage, body fat mass, fat-free mass, body weight, high-sensitivity CRP, total P1NP, BAP measured values ​​and changes from the screening and pre-intake examination after 12 weeks and 24 weeks of intake

[0173] 7. Test results

[0174] 1. Trial Participants

[0175] This study was conducted on healthy Japanese adult men and women who felt weak. Of the 114 subjects who agreed to the study, 56 subjects who met the eligibility criteria were enrolled in the study, and 28 subjects were assigned to the test food group and the placebo group.

[0176] After 24 weeks of intake, there was one participant who did not return to the hospital for the trial (one in the test food group).

[0177] The per protocol set (PPS) excluded one case with no data after allocation (one in the test food group) and seven cases with test product intake rate less than 80% (four in the placebo group and three in the test food group). The safety analysis population (SAF) excluded one case (one in the test food group) in which no safety evaluation items were measured once after allocation. Therefore, the number of cases in each analysis data set was 48 for PPS (24 in the placebo group and 24 in the test food group) and 55 for SAF (28 in the placebo group and 27 in the test food group). The background of the trial participants is shown in the table below.

[0178] [Table 2]

[0179]

[0180] 2. Main results

[0181] The mean (Mean) and standard deviation (SD) of muscle mass, estimated marginal mean (EMM) and its 95% confidence interval (95% CI-, 95% CI+), intergroup difference of EMM and its 95% CI-, 95% CI+, and statistical analysis results are shown in Figure 1 .

[0182] Regarding the measured value of muscle mass after 24 weeks of intake (24w), the test food group showed a significantly higher value compared with the placebo group (P=0.043).

[0183] 3. Secondary Results

[0184] 3-1. Muscle mass (excluding main results)

[0185] The mean and SD of muscle mass, EMM and its 95%CI-, 95%CI+, the inter-group difference of EMM {the inter-group difference of Mean in the screening and pre-intake test (Scr)} and its 95%CI-, 95%CI+, and the statistical analysis results are shown in Figure 1 .

[0186] (1) Measured value

[0187] Among the time points at which a significant difference was seen, the time point at which the test food group showed a higher value than the placebo group was the examination after 12 weeks of ingestion (12w) (P=0.012).

[0188] (2) Change

[0189] Among the time points at which significant differences were observed, the test product group showed higher values ​​than the placebo group at 12 weeks (P=0.012) and 24 weeks (P=0.043).

[0190] 3-2. Physical examination excluding muscle mass, grip strength, 10m walking test, stand-and-go timed test, blood test

[0191] The mean and SD of body measurements excluding muscle mass, grip strength, 10m walk test, timed stand-and-go test, and blood tests, the difference between groups, and 95% CI-, 95% CI+, and the statistical analysis results are shown in Figures 2 to 4 .

[0192] (1) Scr

[0193] Among the items where significant differences were seen, the item in which the test food group showed a lower value than the placebo group was BAP (P=0.045).

[0194] (2) Measured value

[0195] a. 12w

[0196] Among the items with significant differences, the test food group showed lower values ​​than the placebo group in body fat percentage (P=0.019) and fat mass (P=0.031), and showed higher values ​​in fat-free mass (P=0.014).

[0197] b. 24w

[0198] Among the items with significant differences, the test food group showed lower values ​​than the placebo group in body fat percentage (P=0.008) and fat mass (P=0.005), and showed higher values ​​in fat-free mass (P=0.042).

[0199] (3) Change

[0200] a. 12w-Scr

[0201] Among the items with significant differences, the test food group showed lower values ​​than the placebo group in body fat percentage (P=0.019) and fat mass (P=0.031), and showed higher values ​​in fat-free mass (P=0.014).

[0202] b. 24w-Scr

[0203] Among the items with significant differences, the test food group showed lower values ​​than the placebo group in body fat percentage (P=0.008) and fat mass (P=0.005), and showed higher values ​​in fat-free mass (P=0.042).

[0204] 8. Inspection

[0205] The muscle mass after 24 weeks of intake, which was set as the primary outcome, showed significantly higher values ​​in the test food group than in the placebo group ( Figure 1 ), the estimated marginal mean (EMM) and its 95% confidence interval were 36.6kg (36.3 to 36.9) for the test food group, 36.1kg (35.8 to 36.4) for the placebo group, and the difference between the groups was 0.4kg (0.0 to 0.9). In addition, the change in the amount of weight from the screening and pre-intake examination after 24 weeks of intake was also a value significantly higher in the test food group than in the placebo group. The EMM and its 95% confidence interval were 0.5kg (0.1 to 0.8) for the test food group, 0.0kg (-0.3 to 0.3) for the placebo group, and the difference between the groups was 0.4kg (0.0 to 0.9) ( Figure 1 ). In this study, screening and pre-intake examinations and 24-week post-intake examinations were conducted from winter to summer. However, there are reports that fat-free mass (weight of muscle, viscera, etc. minus fat from body weight) changes by 0.3kg to 0.4kg from winter to summer (Ryuji Ikeuchi, Takeshi Morimoto, Hirokiya Nishikawa, Seasonal changes in body composition by bioelectrical impedance method - comparison between the elderly and young -, Nichisei Shoji, 1994; 31(2): 69-73). Furthermore, in a report by Oka et al. (Takuya Oka, Motokai Kato, Factors causing changes in body fat percentage and patterns of changes in human weight and body composition, Kuroshio Science, 2012; 5(2): 161-7), who observed changes in body composition of the same male over a 7-year period, the fluctuation range of skeletal muscle percentage over 7 years was 0.7%. If applied to the muscle mass in this study, it is less than 0.4kg. Based on the above, changes in muscle mass of more than 0.4kg are considered to be clinically significant. In addition, changes of more than 0.4 kg in muscle mass were confirmed after 12 weeks of intake, and both the measured values ​​and the changes in the screening and pre-intake examinations were higher in the test food group than in the placebo group ( Figure 1). In addition, since the difference in EMM between the groups and its 95% confidence interval was 0.6 kg (0.1 to 1.0), the intake of the test food in this study after 12 weeks of intake has clinical significance and is considered to contribute to the increase in muscle mass.

[0206] After 12 weeks and 24 weeks of intake, in addition to muscle mass, body fat percentage, fat mass, and fat-free mass also showed significant differences between the groups ( Figure 3 , 4 ). The body composition of this test was determined by the bioelectrical impedance method (bioelectrical impedance analysis: BIA method) using a body composition meter MC-780A-N33). The fat-free mass is calculated from the height and the resistance value (impedance) of the body, but since most of it is muscle tissue, the muscle mass measured by the BIA method is estimated based on the fat-free mass. Therefore, if the skeletal muscle mass in the body increases, the fat-free mass and muscle mass increase at the same time. On the other hand, the fat mass is calculated by subtracting the fat-free mass from the body weight, but since the effect of the intake of the test food on the body weight was not observed ( Figure 3 , 4 ), so according to the measurement principle of the BIA method, if the fat-free mass increases, the fat mass will inevitably decrease. Based on the above, it is believed that the fat-free mass increases with the increase in skeletal muscle mass caused by the intake of the test food, and the fat mass will inevitably decrease, and as a result, the body fat percentage also decreases, and a significant difference is seen between the groups.

[0207] 9. Conclusion

[0208] Based on the results of these Examples, it was confirmed that the composition of the present invention is effective for increasing or maintaining muscle mass or muscle strength, and is effective for preventing obesity, reducing body fat percentage, reducing body fat mass, or reducing lean mass.

[0209] Industrial Applicability

[0210] According to the present invention, muscle mass or muscle strength can be improved or maintained, and obesity, reduction of body fat percentage, reduction of body fat amount or reduction of fat-free mass can be prevented thereby in addition. In addition, the proteoglycan used in the present invention is from organisms, and toxicity is low. Further, because the proteoglycan used in the present invention can be prepared from conventionally discarded raw materials, the present invention can contribute to the effective utilization of resources, and can also suppress the rise of medical expenses etc.

[0211] This application is based on Japanese Patent Application No. 2022-124661 filed in Japan, the entire contents of which are incorporated herein by reference.

Claims

1. A composition for increasing or maintaining muscle mass or strength, the composition comprising proteoglycan, the proteoglycan being obtained by a proteoglycan preparation method, the preparation method comprising: The method comprises the steps of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant, and recovering the solution after immersion.

2. A composition for preventing obesity, reducing body fat percentage, reducing body fat mass, or reducing fat-free mass, the composition comprising proteoglycan, the proteoglycan being obtained by a proteoglycan preparation method, the preparation method comprising: The method comprises the steps of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant, and recovering the solution after immersion.

3. The composition according to claim 1 or 2, wherein The surfactant is at least one selected from the group consisting of saponin, lecithin, and sucrose fatty acid esters.

4. The composition according to claim 1 or 2, wherein The biological sample containing proteoglycan is at least one selected from the group consisting of cartilage tissue, muscle fiber and skin of fish, mollusk, bird and mammal.

5. The composition according to claim 4, wherein The biological sample containing proteoglycan is at least one selected from the group consisting of cartilage tissues of fish, birds, and mammals.

6. The composition according to claim 1, which is used for increasing or maintaining muscle mass or muscle strength in healthy subjects.

7. The composition according to claim 1 or 2, which is a cosmetic composition, a food composition, or a pharmaceutical composition.

8. A method for increasing or maintaining muscle mass or strength, the method comprising administering to a subject in need of proteoglycan an effective amount of a proteoglycan obtained by a method for preparing a proteoglycan, the method comprising: The invention further comprises a step of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant, and a step of recovering the solution after the immersion.

9. A method for preventing obesity, reducing body fat percentage, reducing body fat mass, or reducing fat-free mass, the method comprising administering an effective amount of proteoglycan to a subject in need of proteoglycan, the proteoglycan being obtained by a proteoglycan preparation method, the preparation method comprising: The invention further comprises a step of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant, and a step of recovering the solution after the immersion.

10. The method according to claim 8 or 9, wherein The surfactant is at least one selected from the group consisting of saponin, lecithin, and sucrose fatty acid esters.

11. The method according to claim 8 or 9, wherein The biological sample containing proteoglycan is at least one selected from the group consisting of cartilage tissue, muscle fiber and skin of fish, mollusk, bird and mammal.

12. The method of claim 11, wherein: The biological sample containing proteoglycan is at least one selected from the group consisting of cartilage tissues of fish, birds, and mammals.

13. The method according to claim 8, which is used to increase or maintain muscle mass or muscle strength in healthy subjects.

14. A proteoglycan for use in increasing or maintaining muscle mass or muscle strength, wherein the proteoglycan is obtained by a method for preparing proteoglycan, the method comprising: The invention further comprises a step of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant, and a step of recovering the solution after the immersion.

15. A proteoglycan for use in preventing obesity, reducing body fat percentage, reducing body fat mass, or reducing fat-free mass, wherein the proteoglycan is obtained by a proteoglycan preparation method, the preparation method comprising: The invention further comprises a step of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant, and a step of recovering the solution after the immersion.

16. The proteoglycan according to claim 14 or 15, wherein The surfactant is at least one selected from the group consisting of saponin, lecithin, and sucrose fatty acid esters.

17. The proteoglycan according to claim 14 or 15, wherein The biological sample containing proteoglycan is at least one selected from the group consisting of cartilage tissue, muscle fiber and skin of fish, mollusk, bird and mammal.

18. The proteoglycan according to claim 17, wherein The biological sample containing proteoglycan is at least one selected from the group consisting of cartilage tissues of fish, birds, and mammals.

19. The proteoglycan according to claim 14, which is used for increasing or maintaining muscle mass or muscle strength in healthy humans.

20. Use of a proteoglycan for increasing or maintaining muscle mass or strength, wherein the proteoglycan is obtained by a proteoglycan preparation method, the preparation method comprising: The invention further comprises a step of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant, and a step of recovering the solution after the immersion.

21. Use of proteoglycan for preventing obesity, reducing body fat percentage, reducing body fat mass, or reducing fat-free mass, wherein the proteoglycan is obtained by a proteoglycan preparation method, the preparation method comprising: The invention further comprises a step of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant, and a step of recovering the solution after the immersion.

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