Agents for maintaining muscle strength

Abstract

To provide an agent for the maintenance of muscle strength or the like for use in human or the like.SOLUTION: An agent for the maintenance of muscle strength contains a cartilage component mixture containing proteoglycan and collagen. A method for producing a cartilage component mixture containing proteoglycan and collagen includes the steps of: immersing cartilage in an aqueous acetic acid solution of 0.03 mass% or more and less than 4 mass% to prepare a cartilage component extract; and recovering the cartilage component from the resultant extract.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an agent (material) for maintaining muscle strength for use in humans and non-human animals. [Background technology]

[0002] In humans, aging and other factors can cause a decrease in muscle mass and strength, including skeletal muscle, and muscle atrophy. A decrease in muscle mass can also lead to a decrease in basal metabolic rate, increasing the likelihood of developing metabolic syndrome. In Japan, which is becoming a super-aging society, maintaining muscle strength is considered important from the perspective of extending healthy lifespans (Non-Patent Document 1).

[0003] Muscle atrophy is broadly divided into disuse muscle atrophy and progressive muscle atrophy. Disuse muscle atrophy is a significant change in skeletal muscle that occurs due to prolonged inactivity, and includes quantitative changes such as a decrease in muscle fiber diameter and qualitative changes such as type transitions at the muscle fiber and muscle protein level. It can be caused by, for example, prolonged bed rest, immobilization with a plaster cast due to fractures, exposure to microgravity (living in outer space, etc.), the aging process, etc. (Patent Document 1).

[0004] For example, in the case of elderly people, muscle atrophy is induced, which increases the risk of fractures due to falls, and many people end up in a state requiring nursing care, such as being bedridden. Exercise such as strength training, weight training, and aerobic exercise is considered to be effective methods for improving this muscle atrophy, but there is a need for methods (methods other than exercise) to prevent and / or improve muscle atrophy in people who require nursing care and are unable to exercise (Non-Patent Document 1).

[0005] Furthermore, it has been shown that obese middle-aged and elderly people who are not used to exercising and diabetic patients (including those with sarcopenic obesity) are likely to experience a decrease in knee extension muscle strength and a decrease in lean mass of the lower limbs. For these patients, there is a need for methods (other than exercise) to prevent and / or improve muscle atrophy (Non-Patent Document 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-62068 [Non-patent literature]

[0007] [Non-Patent Document 1] Jpn J Rehabil Med 2007; 44: 144-170 [Non-patent document 2] Japanese Journal of Geriatrics 2014;51:99-108 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide a material (agent) for maintaining muscle strength, which can be used by humans and the like. [Means for solving the problem]

[0009] The present invention has been made to solve the above-mentioned problems, and the inventor invented the present invention as a result of searching for substances (such as those extracted from natural products) that have the effect of maintaining muscle strength.

[0010] The present invention includes the following embodiments. (1) An agent for maintaining muscle strength, comprising a cartilage component mixture including proteoglycan and collagen. (2) An oral composition containing the agent according to (1). (3) The oral composition according to (2), wherein the cartilage component mixture is ingested by an adult human being daily, preferably at a dose of 0.5 mg / day to 50 mg / day. (4) A method for producing a cartilage component mixture containing proteoglycan and collagen, comprising the steps of: immersing cartilage in an aqueous solution of acetic acid having a concentration of 0.03% by mass or more and less than 4% by mass to obtain a cartilage component extract; and recovering cartilage components from the obtained extract. (5) The method according to (4), wherein the concentration of the aqueous acetic acid solution is 0.5% by mass or more and 3% by mass or less. (6) The manufacturing method described in (6), wherein the collagen content is at least 20% by mass in terms of solid content. (7) The method for producing the proteoglycan, wherein the molecular weight of the proteoglycan is 400,000 to 650,000. [Effects of the Invention]

[0011] The agent of the present invention can be used to maintain muscle strength in humans and the like. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a process diagram showing an example of the production of a cartilage component mixture. DETAILED DESCRIPTION OF THE INVENTION

[0013] Next, embodiments of the present invention will be described with reference to the drawings. Note that the embodiments described below do not limit the invention according to the claims, and not all of the elements and combinations thereof described in the embodiments are necessarily essential to the solution of the present invention.

[0014] (cartilage component mixture) The product obtained by the manufacturing method of the present invention is a mixture containing proteoglycans as well as other cartilage components. These cartilage components other than proteoglycans include, but are not limited to, collagen and hyaluronic acid. Cartilage is a type of connective tissue present in vertebrates' noses, ribs, joints, around the trachea, ear shells, intervertebral discs, etc., and refers to a complex of extracellular matrix and chondrocytes. The extracellular matrix in cartilage is sometimes referred to as the cartilage matrix. The main components of the cartilage matrix include collagen, chondroitin sulfate, hyaluronic acid, and proteoglycans.

[0015] Proteoglycans (hereinafter sometimes referred to as "PG") are complex polysaccharides consisting of a protein core to which glycosaminoglycans such as chondroitin sulfate and dermatan sulfate are covalently bonded, and are found in large quantities in animal tissues, particularly cartilage tissue. Proteoglycans are also known to exist in vivo in structures in which the core protein is further bound to hyaluronic acid, and their molecular weights are large, ranging from tens of thousands to tens of millions. A typical cartilage-derived proteoglycan is called aggrecan.

[0016] Collagen has a helical structure consisting of three polypeptide molecules with a molecular weight of approximately 100,000, each of which is a chain of amino acids, forming a fibrous or membrane-like structure. The types and number of amino acids that make up collagen are highly distinctive, and one of these features is that it contains amino acids such as hydroxyproline and hydroxylysine, which are not included in the 20 basic amino acids that make up common proteins. These amino acids are unique amino acids found only in collagen and a limited number of closely related proteins, and hydroxyproline in particular accounts for approximately 10% of all amino acids in collagen. For this reason, hydroxyproline can be considered an indicator of collagen content. While there are no particular limitations on the type of collagen based on amino acid composition, type II collagen is preferred as it is found in large amounts in cartilage.

[0017] Hyaluronic acid is a mucopolysaccharide polymer compound with a chain structure consisting of disaccharide units formed by the bond between N-acetylglucosamine and glucuronic acid. Other cartilage components include laminin, fibronectin, and elastin.

[0018] (Example of production of cartilage component mixture) A method (production example) for producing a cartilage component mixture used in the present invention is, for example, a method for extracting cartilage components using a low-concentration aqueous acetic acid solution. Figure 1 is a process diagram showing a typical embodiment of this production example. This production example includes an extraction step (S10) in which frozen cartilage is immersed in an aqueous acetic acid solution to obtain a cartilage component extract, and a recovery step (S20) in which the cartilage components are recovered from the obtained extract.

[0019] The lower limit of the concentration of the aqueous acetic acid solution used in the extraction step (S10) may be 0.03% by mass or more, preferably 0.05% by mass or more, more preferably 0.1% by mass or more, and even more preferably 0.25% by mass or more, from the viewpoint of increasing the extraction efficiency of proteoglycans, etc. On the other hand, the upper limit of the acetic acid concentration in the aqueous acetic acid solution may be less than 4% by mass, preferably 3.5% by mass or less, more preferably 3.1% by mass or less, and even more preferably 2% by mass or less, from the viewpoint of increasing the extraction efficiency of collagen extracted at the same time.

[0020] More specifically, the recovery step (S20) includes a solid-liquid separation step (S21) for removing remaining cartilage, a degreasing step (S22) for removing lipids and the like from the recovered extract, a filtration step (S23), a purification step (S24), and a drying step (S25).

[0021] In the degreasing step (S22), powdered cellulose and / or an oil-absorbing mat are used to easily adsorb and remove components such as lipids that may be present in the proteoglycan extract. In the filtration step (S23), a conventional method using filter paper or the like is used to obtain an extract after removing lipids and other contaminants. Fine mesh or ultrafiltration membranes may also be used. For example, the extract is recovered by solid-liquid separation using a separation membrane with an appropriate molecular weight cutoff. After removing insoluble matter using a conventional method such as a magnetic trap, the resulting filtrate may be solidified in a vacuum freeze dryer in the drying step (S25). Alternatively, it can be dried using a spray dryer to obtain a powdered solid.

[0022] The molecular weight of the proteoglycan in the cartilage component mixture containing proteoglycan obtained by this second embodiment is preferably 400,000 to 650,000.

[0023] The daily intake amount of the cartilage component mixture used in the present invention (when taken as an oral composition) can be adjusted as appropriate depending on the intake form, purpose of use, age, body weight, etc., but in order to achieve the desired effect (such as the effect of inhibiting muscle atrophy), the daily intake amount for an adult human is preferably 0.1 mg / kg / day or more, more preferably 0.5 mg / kg / day or more, more preferably 1 mg / kg / day or more, more preferably 2 mg / kg / day or more, more preferably 3 mg / kg / day or more, more preferably 4 mg / kg / day or more, and even more preferably 5 mg / kg / day or more, and from the perspective of safety in humans when taking the composition, the daily intake amount is preferably 10 mg / kg / day or less, more preferably 8 mg / kg / day or less, and even more preferably 6 mg / kg / day or less.

[0024] (Agent for maintaining muscle strength) The form of the agent for maintaining muscle strength according to the present invention is not particularly limited, but examples thereof include liquid, solid, and powder forms.

[0025] (Maintaining muscle strength) Muscle strength maintenance includes, for example, suppression of muscle atrophy, and increase and / or maintenance of muscle mass.

[0026] (Oral Composition) The oral composition according to the present invention is, for example, a food or drink (including functional foods, foods for specified health uses, supplements, etc.), a pharmaceutical product, or the like.

[0027] For example, when the oral composition is a food or beverage, the food or beverage may take the form of various foods and beverages such as breads, cakes, noodles, confectioneries, jellies, frozen foods, ice cream, dairy products, beverages, etc., as well as forms similar to those of the oral administration preparations described above (tablets, capsules, syrups, etc.) Foods in various forms can be prepared by using the active ingredient of the present invention alone or in appropriate combination with other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, flavorings, stabilizers, colorants, antioxidants, moisturizers, thickeners, etc.

[0028] For example, when the oral composition is a pharmaceutical, the pharmaceutical is generally easy to formulate a convenient daily dosage regimen that can be adjusted according to the degree of pain, and the pharmaceutical form can be, for example, a solid form or a liquid form.The solid form can include, for example, powder, tablets, pills, capsules, cachets, lozenges, suppositories, and dispersible granules.For example, in powders, the carrier is generally a finely divided solid that is mixed with a finely divided active ingredient.For example, in tablets, the active ingredient is generally mixed with a carrier having the necessary binding capacity in an appropriate ratio and formed into the desired shape and size.Suitable carriers can include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting wax, cocoa butter, etc. The pharmaceutical may also contain ingredients such as excipients, stabilizers, preservatives, binders, disintegrants, hydrocarbons, fatty acids, alcohols, esters, pH adjusters, and preservatives as needed, within the range that does not impair the desired effects.

[0029] Muscle atrophy is the loss of muscle mass. Muscle atrophy also results in a loss of muscle strength. Muscle atrophy can be caused by a disease of the muscle itself (myopathic muscular atrophy) or by damage to the motor nerves that directly transmit motor commands to the muscle (neuropathic muscular atrophy). Generally, muscle diseases tend to atrophy the muscles from the shoulders to the upper arms and the waist to the thighs (proximal muscles), while nerve diseases tend to atrophy the muscles in the extremities (distal muscles of the limbs). Typical muscle diseases include hereditary muscle disorders such as muscular dystrophy and inflammatory muscle disorders such as polymyositis and dermatomyositis. Neuropathic muscular atrophy is often caused by peripheral nerve disorders (neuropathy), which can have a variety of causes, including trauma, compression, inflammatory disorders, and hereditary causes.

[0030] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the following examples, the unit % used to indicate the content of components contained in the agent of the present invention means % by mass. [Example]

[0031] Examples of the present invention will be described below.

[0032] Examples 1 to 8: Production of powder mixture of proteoglycan and collagen (cartilage component mixture) 400 g of nasal cartilage extracted from the head of a chum salmon that had been frozen and stored at -30°C to -20°C was prepared as the starting material. 2000 mL of aqueous acetic acid solutions of various concentrations according to Comparative Example 1 and Examples 1 to 8 were added to this, and extraction was carried out at an extraction temperature of 30°C to 40°C for 48 to 72 hours. The various concentrations (%), extraction temperatures (°C), and extraction times (hr) according to Comparative Example 1 and Examples 1 to 8 are as shown in Table 1.

[0033] This extract was filtered through No. 26 filter paper (110 mm) to remove insoluble matter. Next, 2% powdered cellulose (manufactured by Nippon Paper Industries Co., Ltd., product name "KC Flock W-400G") was added to the liquid volume, and the mixture was stirred for 30 minutes and then filtered. The filtrate was concentrated using a hollow fiber membrane with a molecular weight cutoff of 50,000 until the liquid volume was reduced to 1 / 10. Further concentration and purification were repeated while diluting with water, ultimately yielding 500 to 800 g of a concentrated liquid (pH 6 to 7). The resulting concentrated liquid was then freeze-dried to obtain 10 to 20 g of a mixture of proteoglycan and collagen (lyophilized product).

[0034] The freeze-dried product was measured for the following items, and the results are shown in Table 1 below. PG yield (%): Value (%) calculated by ((proteoglycan content in the obtained lyophilized product) / (weight of the nasal cartilage 400 (g))) x 100 ·PG molecular weight (×10 4 ): Proteoglycan molecular weight. For example, the entry "56" in Example 1 of Table 1 indicates a molecular weight of 560,000 (560,000). PG content ratio: the proteoglycan content in the freeze-dried product of each Example is a relative value (relative value shown in comparison with Comparative Example 1), with the proteoglycan content in the freeze-dried product of Comparative Example 1 being set at 100. Collagen content (%): Collagen content in the lyophilized product - "-": Indicates that no measurement has been performed.

[0035] (Method for quantifying proteoglycan (PG)) Approximately 1 g of the dried product obtained by the production methods of Comparative Example 1 and Examples 1 to 8 was precisely weighed and diluted to exactly 10 mL with phosphate buffer (pH 6.8) to prepare a sample solution. Each sample solution was passed through a 0.45 μm membrane filter and then subjected to HPLC under the following operating conditions. The amount of proteoglycan was determined from a calibration curve of the standard. The calibration curve was prepared by drying a proteoglycan standard (derived from salmon nasal cartilage, Fujifilm Wako Pure Chemical Industries, Ltd., 162-22131) in a vacuum desiccator (silica gel) at room temperature for 3 hours, then precisely weighing the sample and dissolving it in the same phosphate buffer as the sample to prepare a standard solution for the calibration curve. Furthermore, the molecular weight of the peak top was determined from a calibration curve prepared using Shodex STANDARD P-82 (Showa Denko K.K.) as a molecular weight marker.

[0036] Operating conditions Analyzer: HPLC analyzer Detector: Differential refractive index detector (RID-10A, Shimadzu Corporation) Column: Gel filtration column (TSKgel G5000PWXL manufactured by Tosoh Corporation) Column temperature: 40℃ Sample injection volume: 50 μL Mobile phase: Phosphate buffer (pH 6.8) Flow rate: 0.5mL / min

[0037] (Collagen quantification method) Collagen is characterized by containing hydroxyproline and hydroxylysine, which are not found in general proteins. Hydroxyproline is said to account for approximately 10% of all amino acids in collagen, and the collagen content can be estimated by quantifying this hydroxyproline (Leather Science, Vol. 56, No. 2, pp. 71-79, 2010, "Functionality of Natural Collagen"). The collagen content of dried product samples obtained by the production methods of Comparative Example 1 and Examples 1 to 8 was calculated by measuring the amount of hydroxyproline.

[0038] [Table 1]

[0039] As shown in Table 1, it was confirmed that the yield of proteoglycan was improved in Examples 1 to 8 compared to Comparative Example 1. [Experiment 1: Evaluation of muscle strength maintenance] Using laboratory animals (mice), an experiment was carried out to evaluate the muscle atrophy inhibitory effect of the oral composition containing the cartilage component mixture of Example 4. This evaluation was carried out by measuring the weight of the gastrocnemius and soleus muscles.

[0040] The experimental method is described below. First, 20 7-week-old male DDY mice (Kiwa Laboratory Animal Research Institute) were obtained. The obtained mice were kept in a normal condition (with normal food and water provided ad libitum) for one week.

[0041] After the one week of normal breeding, the following two experimental groups were set up, each consisting of 10 animals, and the animals were kept under the following conditions (regular food and drinking water) for four weeks (28 days): Group 1 was further orally administered daily using a probe under the following conditions: Control group: A group that received only drinking water. Group 1: A group of mice given 66.67 mg / kg / day of the cartilage component mixture of Example 4. When converted to an adult human dose based on a human equivalent dose (HED) of 12.3 in mice (see the document "Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers"), giving 66.67 mg / kg / day to mice corresponds to giving 5.42 mg / kg / day of the cartilage component mixture to an adult human (60 kg).

[0042] After 7 weeks of breeding, the mice were weighed, and the gastrocnemius (muscle located outside the soleus) and soleus muscles were removed from each group of mice under anesthesia. The weights of the removed gastrocnemius and soleus muscles were measured.

[0043] The results of the measurements are shown in Table 2 below. The measurements were performed on the gastrocnemius and soleus muscles (left and right) of one animal in each group (10 animals). The results below are the average total weight (g) of the left and right muscles per animal in each group. No changes in body weight were observed in any of the groups. However, an increase in gastrocnemius muscle weight (p<0.01, significant difference determined by Student's t-test) and soleus muscle weight was observed in Group 1 compared to the control group.

[0044] [Table 2]

[0045] The above has described the embodiments (including examples) of the present invention with reference to the drawings, but the specific configuration of the present invention is not limited to this, and even if there are design changes, etc., within the scope that does not deviate from the gist of the present invention, they are included in the present invention. [Industrial Applicability]

[0046] The present invention can provide an agent for maintaining muscle strength for use in humans, etc.

Claims

1. A method for producing a cartilage component mixture comprising proteoglycan and collagen, wherein the proteoglycan has a molecular weight of 400,000 to 650,000, the method comprising the steps of: immersing cartilage in an aqueous solution of acetic acid having a concentration of 0.03% by mass or more and less than 4% by mass to obtain a cartilage component extract; and recovering the cartilage component from the obtained extract.

2. A method for manufacturing a mixture of cartilage components for maintaining muscle strength, as described in claim 1.

3. 2. The method according to claim 1, wherein the concentration of the aqueous acetic acid solution is 0.5% by mass or more and 3% by mass or less. End

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