JPWO2019077810A1 - Periostin and PKM2 secretagogue - Google Patents

Abstract

Provided is an agent or composition useful for muscle cell proliferation and the like. The agent or composition is composed of at least one component (A) selected from acteosides, echinacosides and salts thereof, and at least one component (B) selected from periostin and PKM2.

Description

The present invention relates to an agent or composition capable of promoting (increasing) the secretion (expression) of periostin and PKM2.

In spinal cord injury, axons rupture in the traumatically crushed or transected spinal cord, resulting in dysfunction of the injured spinal cord and the movement and sensation of the trunk and upper and lower limbs controlled by the lower spinal cord.

The clinical treatment for spinal cord injury was to administer a large amount of steroids immediately after the injury, but its effectiveness for functional recovery has been questioned (Non-Patent Document 1), and it is recommended because of the high risk of side effects. The current situation is that it has not. In relatively mild injuries, rehabilitation may improve residual function, but in "chronic" patients months-years after injury, function The chances of recovery are extremely low.

In recent years, regenerative medicine applying neural stem cells and iPS cells has been seen as a promising next-generation treatment strategy for spinal cord injury, and basic research is being vigorously pursued (Non-Patent Documents 2 and 3), but nerves in mice. In stem / progenitor cell transplantation, axillary extension and recovery of motor function are observed in the injured area up to 9 days after injury (subacute phase), whereas transplantation 42 days after injury (chronic phase) Is completely invalid (Non-Patent Document 4).

Transplantation of nerve stem / progenitor cells and simultaneous treatment of neurotrophic factors (Non-Patent Document 5) or simultaneous treatment of ChABC * (* axon inhibitor secreted from glial scars) for animals with SCI An enzyme that decomposes chondroitin sulfate proteoglycan) (Non-Patent Document 6) or simultaneous treatment of exercise load by walking with a tread mill (Non-Patent Document 7) has been tried, but the degree of functional recovery is still limited. In other words, it is still extremely difficult to recover the function of spinal cord injury in the chronic phase after injury.

Lakartidningen (2005) 102, 1887-1888. PLoS One (2009) 4, e7706; PNAS (2010) 107, 12704-12709. Cell Res (2013) 23, 70-80. Molecular Brain (2013) 6, 3 BBRC (2010) 393, 812-817. J Neurosci (2010) 30, 1657-1676. Sci Rep (2016) 6, 30898

An object of the present invention is to provide an agent or composition capable of promoting the secretion of periostin or PKM2 (pyruvate kinase M2, pyruvate kinase M2 isozyme).

Another object of the present invention is to provide an agent or composition capable of extending (elongating) axons and improving (improving) myocyte proliferation and muscular atrophy.

Another object of the present invention is to provide an agent or composition useful for improving various symptoms / diseases and improving (improving) / maintaining motor function and physical strength.

As a result of diligent studies, the present inventors have found that specific components (acteosides and echinacosides) promote the secretion of periostin and PKM2, and surprisingly, through the secretion of such periostin and PKM2. The present invention was completed by finding that signals are diffused systemically, muscle cells are proliferated, muscle mass is increased, and axon (neurite) extension (extension) is promoted. ..

That is, the present invention relates to the following inventions and the like.
[1]
An agent or composition containing at least one component (A) selected from acteosides, echinacosides and salts thereof, and for promoting the secretion of periostin and / or pyruvate kinase M2.
[2]
Contains at least one component (A) selected from acteosides, echinacosides and salts thereof, for the growth of muscle cells, the increase of muscle (or muscle mass, the same applies hereinafter) and / or the improvement (treatment) of muscle atrophy. Agent or composition.
[3]
Further, the agent or composition according to [2] for extending and / or repairing an axon.
[4]
Containing at least one component (B) [and at least one component (A) selected from acteosides, echinacoside and salts thereof] selected from periostin and PKM2, axon extension, axon repair, An agent or composition for improving (treating) muscle cell proliferation, muscle gain, and / or muscle atrophy.
[5]
The agent or composition according to [4], wherein the component (B) is present in vitro.
[6]
The agent or composition according to any one of [1] to [5] for use in at least one application selected from the following (1) to (6).
(1) Treatment and / or improvement of neurological disorders (2) Treatment and / or improvement of muscle disorders (3) Treatment and / or improvement of sarcopenia (4) Treatment and / or improvement of locomotive syndrome (5) Treatment and improvement of paralysis / Or improvement (6) Improvement (improvement) and / or maintenance of motor function and / or physical strength [7]
The agent or composition according to [6], wherein neurological disease, muscle disease, sarcopenia, locomotive syndrome and paralysis are symptoms of the chronic phase.
[8]
The agent or composition according to any one of [1] to [7] for treating and / or ameliorating spinal cord injury in the chronic phase.
[9]
The agent or composition according to any one of [1] to [3] and [6] to [8], which contains at least an acteoside as the component (A).
[10]
The agent or composition according to any one of [1] to [3] and [6] to [9], which contains a syrup component as a component containing the component (A).
[11]
The agent or composition according to any one of [1] to [10] for intramuscular injection or oral use.
[12]
The agent or composition according to any one of [1] to [11], which is a food or drink.
[13]
The agent or composition according to any one of [1] to [12], which is a supplement, a health food, a food with a functional claim, a food with a nutritional function, or a food for specified health use.
[14]
A method for promoting the secretion of periostin and / or pyruvate kinase M2 by using at least one component (A) [or an agent or composition containing the component (A)] selected from acteosides, echinacosides and salts thereof. ..
[15]
Using at least one component (A) [or an agent or composition containing component (A)] selected from acteosides, echinacosides and salts thereof, muscle cells are proliferated, muscles are increased (increased) and / Or a method of improving (treating) muscle atrophy.
[16]
[15] The method of [15], wherein the axon is extended and / or repaired.
[17]
Axons are squeezed using at least one component (B) selected from periostin and PKM2 [and an agent or composition containing at least one component (A) selected from acteosides, echinacoside and salts thereof]. A method of extending, repairing axons, proliferating muscle cells, gaining muscle, and / or improving (treating) muscle atrophy.
[18]
The method according to [17], wherein the component (B) is present outside the living body.
[19]
The method according to any one of [14] to [18], which comprises at least one selected from the following (1) to (6).
(1) Treat and / or improve neurological disorders (2) Treat and / or improve muscle disorders (3) Treat and / or improve sarcopenia (4) Treat and / or improve locomotive syndrome (5) Treat and / or improve paralysis (6) Improve (improve) and / or maintain motor function and / or physical fitness [20]
The method according to [19], wherein neurological disease, muscle disease, sarcopenia, locomotive syndrome and paralysis are symptoms of the chronic phase.
[21]
The method according to any of [14] to [20], which treats and / or improves spinal cord injury in the chronic phase.
[22]
The method according to any one of [14] to [21], which comprises at least an acteoside as the component (A).
[23]
The method according to any one of [14] to [22], which contains a Nikujuyo component as a component containing the component (A).
[24]
The method according to any one of [14] to [23], which is administered intramuscularly or orally.
[25]
The method according to any one of [14] to [24], wherein the agent or composition is a food or drink.
[26]
The method according to any one of [14] to [25], wherein the agent or composition is a supplement, a health food, a food with a functional claim, a food with a nutritional function, or a food for specified health use.

In the present invention, the secretion of periostin and PKM2 can be promoted. Then, the present inventors have confirmed that periostin and PKM2 have a proliferative action on muscle cells and an axon extension action on nerve cells. Therefore, it is useful for the treatment and / or improvement (improvement) of various diseases / symptoms related to these functions.

In another aspect of the present invention, muscle cell proliferation (or promotion of muscle cell proliferation or increase in muscle mass) and muscle atrophy can be improved (improved).

Therefore, the present invention relates to diseases / symptoms caused or related to muscle cell depletion, muscle atrophy, etc. [for example, neurological disease (spinal cord injury, etc.), muscle disease, sarcopenia, locomotive syndrome, paralysis (eg, hand and / or foot). It can be suitably applied for the treatment or improvement of these diseases / symptoms, such as paralysis of limbs, paralysis of limbs), especially in the chronic phase.

In addition, in order to promote the proliferation of muscle cells, it is possible to improve or maintain motor function and physical strength regardless of whether or not any symptom or disease has occurred.

In addition, such proliferation of muscle cells may be carried out through promotion of secretion of periostin and PKM2 (pyruvate kinase M2).

A in FIG. 1 is the axon density when the culture supernatant obtained by treating the skeletal muscle cells with Nikujuyo extract is treated on the nerve cells, and B in FIG. 1 is obtained by treating the skeletal muscle cells with acteoside. It is a graph which shows the axon density when the culture supernatant was treated with the nerve cell. FIG. 2A is a graph showing the axon density when the nerve cells are treated with Nikujuyo extract, and FIG. 2B is a graph showing the axon density when the nerve cells are treated with acteoside. FIG. 3 is a graph showing the cell viability when skeletal muscle cells are treated with Nikujuyo extract or acteoside. FIG. 4 shows the hind limbs when Nikujuyo extract was intramuscularly injected into chronic spinal cord injured mice prepared by crushing the spinal cord on the Basso mouse scale (A in FIG. 4) and the Toyama mouse scale (B in FIG. 4). It is a graph which shows the transition of the motor function (administration period 35 days). FIG. 5 shows hindlimb motor function when acteoside is intramuscularly injected into chronic spinal cord injured mice prepared by crushing the spinal cord on the Basso mouse scale (A in FIG. 5) and the Toyama mouse scale (B in FIG. 5). It is a graph which shows the transition (administration period 35 days). FIG. 6 shows administration of Nikujuyo extract by intramuscular injection into chronic spinal cord injured mice prepared by completely cutting the spinal cord on the Basso mouse scale (A in FIG. 6) and the Toyama mouse scale (B in FIG. 6). It is a graph which shows the transition of the hind limb motor function during the period (administration period 56 days). FIG. 7 is a graph showing the wet weight of the gastrocnemius muscle when the biceps femoris extract is intramuscularly injected into the biceps femoris of the hind limbs of a mouse with chronic spinal cord injury prepared by completely cutting the spinal cord. FIG. 8 shows the hind limbs when Nikujuyo extract was orally administered to chronic spinal cord injured mice prepared by crushing the spinal cord on the Basso mouse scale (A in FIG. 8) and the Toyama mouse scale (B in FIG. 8). It is a graph which shows the transition of motor function. FIG. 9 shows the wet weight of hindlimb skeletal muscles (biceps femoris, vastus lateralis, gastrocnemius, tibialis anterior muscles) when Nikujuyo extract was orally administered to chronic spinal cord injured mice prepared by crushing the spinal cord. It is a graph which shows the ratio (muscle wet weight / body weight) to the body weight. FIG. 10 shows the results of identifying proteins that increase in the culture supernatant from acteoside-treated myocytes. FIG. 11 is a graph showing axon density when nerve cells are treated with recombinant periostin (A in FIG. 11) or recombinant PKM2 (B in FIG. 11). FIG. 12 is a graph showing cell viability when skeletal muscle cells are treated with recombinant periostin (A in FIG. 12) or recombinant PKM2 (B in FIG. 12). FIG. 13 shows hindlimb motor function when acteoside is intramuscularly injected into chronic spinal cord injured mice prepared by crushing the spinal cord on the Basso mouse scale (A in FIG. 13) and the Toyama mouse scale (B in FIG. 13). It is a graph which shows the transition of. FIG. 14 shows the wet weight of the hindlimb skeletal muscles (biceps femoris, vastus lateralis, gastrocnemius, tibialis anterior) when acteoside was intramuscularly injected into a chronic spinal cord injured mouse prepared by crushing the spinal cord. It is a graph which shows the ratio (muscular wet weight / body weight). FIG. 15 shows the results of quantifying the area of presynapses projected onto motor nerve cells when acteoside is intramuscularly injected into chronic spinal cord injured mice prepared by crushing the spinal cord. FIG. 16 is a graph showing the evaluation results of motor function by a beam test when the sarcopenia mouse model was orally administered with Nikujuyo extract.

<Agent / Composition>
The agent or composition of the present invention contains a specific component (A) [and / or component (B)] and can be used in various applications.

[Use]
The agent or composition of the present invention (the agent or composition of the first aspect) is useful for promoting the secretion of periostin and / or pyruvate kinase M2 (PKM2, pyruvate kinase M2 isozyme). The periostin and / or PKM2 is not particularly limited, but may be secreted from muscles (skeletal muscles and the like) or muscle cells.

Then, according to the study by the present inventors, it was confirmed that the secretion of periostin and PKM2 (via secretion) causes the proliferation of muscle cells, the increase in muscle mass, and the extension or repair of axons. doing.

Therefore, the agent or composition of the first aspect can be applied to the improvement / improvement of symptoms / diseases related to muscle cell proliferation, muscle mass increase (increase) and axon extension, as described later. ..

The agent or composition of the second aspect is useful for myocyte proliferation (promotion of proliferation), increase in muscle mass and / or improvement of muscle atrophy (treatment).

In muscle cells and muscular atrophy, the target muscles are not particularly limited, but may include at least skeletal muscles, particularly skeletal muscles that are easily directly linked to motor function, for example, muscles of hands and feet.

The agent or composition of such a second aspect may have an axon extension and / or repair function. Specifically, the agent or composition of the second aspect may be used for repair or extension (elongation) of neurites [particularly atrophied or defective neurites (axons and dendrites)].

As described above, the agent or composition of the second aspect has a muscle cell proliferation function, a muscle mass increasing function, and a muscular atrophy improving function (further, an axon extension / repair function). Therefore, it can be applied to various uses in relation to such a function.

For example, the agent or composition of the second aspect may be used for at least one application selected from the following (1) to (5).

(1) Treatment and / or improvement of neurological disorders (2) Treatment and / or improvement of muscle disorders (3) Treatment and / or improvement of sarcopenia (4) Treatment and / or improvement of locomotive syndrome (5) Paralysis (hands and hands and / Or treatment and / or improvement of foot paralysis, limb paralysis, etc.)

Neurological disorders include, for example, spinal cord injury, amyotrophic lateral sclerosis (ALS), and the like.

The myopathic disease may be a myopathic disease (myopathy), a neurogenic disease (neuropathy), or the like, and may be a myogenic disease in particular.

Specific muscle diseases include, for example, muscular atrophy [eg, myogenic muscular dystrophy (eg, muscular dystrophy), neurogenic muscular atrophy (eg, spinal muscular atrophy (SMA), bulbar muscular muscle). Atrophy, etc.)], diseases with abnormal muscle cells (eg, muscular dystrophy, muscular rigidity), diseases with abnormal energy metabolism (eg, mitochondrial encephalomyopathy, periodic limb palsy, etc.), neuromuscular junction Partial diseases (eg, severe muscular dystrophy, Lambert-Eaton syndrome, etc.) and the like can be mentioned.

The muscle disease (muscle atrophy) also includes those associated with a neurological disease (for example, muscle disease or muscle atrophy associated with the neurological disease such as muscle atrophy due to spinal cord injury).

The factors (causes) of sarcopenia, locomotive syndrome and paralysis are not particularly limited, and may be due to the above-mentioned neurological diseases (spinal cord injury, etc.) and muscle diseases (myogenic diseases, etc.), and are not based on these. There may be.

Furthermore, the agent or composition of the second aspect promotes the proliferation of muscle cells and the increase of muscle mass (furthermore, the extension of axons), so that the motor function and the motor function may be affected regardless of whether or not any symptom / disease is reached. Can improve or maintain physical fitness. Therefore, the agent or composition of the second aspect may be used for (6) improvement (improvement) and / or maintenance of motor function and / or physical strength.

For example, the agent or composition of the second aspect improves (improves) the motor function and / or physical strength of a subject (human) having a specific disease / symptom as described in (i) above (1) to (4). And / or may be used to maintain (ii) improve (improve) and / or physical fitness of a subject (human) with a disease / symptom other than (1) to (4) above. It may be used to maintain (iii) to improve (improve) and / or maintain the motor function and / or physical strength of a subject (human) who does not have any disease / symptom (for example, physical strength associated with aging, etc.) It may be used to improve and / improve the decrease.

As described in the first aspect above, the component (A) realizes the proliferation of muscle cells, the increase in muscle mass, and the extension of axons by secreting periostin and PKM2 (via secretion). it can.

Therefore, in the second aspect, muscle cell proliferation (promotion of proliferation), increase in muscle mass and / or improvement of muscular atrophy [Furthermore, the above-mentioned various uses related to such functions ((1) to (1) to ( 6) etc.)] may be performed through (via) the secretion of periostin and / or PKM2.

The agents or compositions of the present invention may ameliorate various symptoms or diseases as described above, but such symptoms or diseases (eg, neurological disorders, muscle disorders, sarcopenia, locomotive syndrome, paralysis, etc.) It may be in the chronic phase (chronic disease, chronic symptom). The chronic phase is not particularly limited and depends on the symptom or disease, but usually refers to the state after the lapse of the variable phase (acute phase, severe symptom). For example, in the case of spinal cord injury (in the case of humans), it is often the chronic phase several months after the serious injury.

It is usually difficult to treat or ameliorate chronic symptoms, but according to the agents or compositions of the invention, even chronic diseases or symptoms (eg, spinal cord injury, muscle disease associated with spinal cord injury) Can be treated and / or improved efficiently.

[Component (A)]
The agent or composition of the present invention (first aspect and second aspect) contains at least one component (A) selected from acteosides, echinacosides and salts thereof (particularly pharmaceutically acceptable salts). Including.

The salt is not particularly limited, and for example, a metal salt [for example, an alkali metal salt (for example, lithium salt, sodium salt, potassium salt, etc.), an alkaline earth metal salt (for example, magnesium salt, calcium salt, etc.), a periodic table. Group 13 metal salts (eg aluminum salts), transition metal salts (eg salts such as zinc)], ammonium salts, amine salts [eg alkylamine salts (eg trimethylamine salts, trialkylamine salts of triethylamine salts) ), Alkanolamine salt (for example, monoethanolamine salt, triethanolamine salt), cyclic amine salt (for example, pyridine salt)] and the like.

In particular, the component (A) may contain at least acteoside [Acteoside, Verbascoside, Kusaginin].

When the component (A) contains an acteoside and an echinacoside, the ratio thereof is not particularly limited, but for example, acteoside / echinakoside (mass ratio) = 100/1 to 0.01/1, preferably 50/1. Even if it is about 0.02 / 1 (for example, 30/1 to 0.03 / 1), more preferably 20/1 to 0.05 / 1 (for example, 15/1 to 0.07 / 1). Often, 10 / 1-0.1 / 1 (eg, 1/1-0.1 / 1, 0.9 / 1-0.2 / 1, 0.8 / 1-0.25 / 1, 0. 7 / 1-0.3 / 1 etc.).

The component (A) may be a synthetic product or may be derived from a natural product. As the synthesis method, a conventional method can be used.

For example, acteosides are found in plants of the family Orobanche coeruleus (eg, Nikjuyo (meat servant)), dioe, olives, ribwort plantain, etc., and may be obtained from such plants.

Echinacoside is a plant of the Echinacea family (for example, Echinacea angustifolia), Echinacea angustifolia, Echinacea pallida, Echinacea purpurea, etc. ing.

Plants of the family Broomrape, especially Nikujuyou, often contain other components in addition to acteosides (and echinacosides), and in combination with having these components, the effects of the present invention are effectively effective. Easy to develop. Further, in terms of safety, Nikujuyo and the like can be preferably used.

Therefore, the agent or composition of the present invention may contain, as a component containing the component (A), a component of a broomrape family plant such as a component of Orobanche coeruleus.

Examples of the components of the broomrape family plant include broomrape family plants and their extracts (extracts), and these may be combined. Specifically, examples of the Nikujuyo component include Nikujuyo, an extract of Nikujuyo (Nikujuyo extract), and the like, and these may be combined.

Plants of the family Broomrape may use parts such as roots, stems, trunks, bark, leaves, flowers, fruits, pericarps, spikes, seeds, seed coats, etc., or may be used in combination thereof, and the whole plant may be used. You may use it. In particular, at least stems (fleshy stems) may be preferably used.

The cistanche salsa is not particularly limited, and examples thereof include cistanche salsa, cistanche deserticola and / or cistanche tubulosa stalks (flesh stalks).
Among these, Kankanikujuyo may be preferably used.

As the extract, a commercially available product may be used, and a plant of the family Broomrape (Nikujuyou, etc.) is extracted by a conventional method {for example, an extraction solvent [for example, water, alcohol (methanol, ethanol, etc.), a mixture thereof, etc. ], It can also be obtained by extraction treatment at room temperature or heating by a conventional method or a method similar thereto}.

Among the extracts, a portion having a particularly high content or concentration of the component (A) may be separated or fractionated and used.

At the time of extraction, a plant of the family Broomrape (such as Broomrape) may be used as it is or crushed (crushed). In addition, the extract may be further subjected to concentration and drying (normal temperature drying, freeze drying, etc.).

The form of the component of the plant of the family Broomrape (such as the component of Orobanche coeruleus) is not particularly limited, but may be powdery (or powdery or granular), for example.

[Form / administration]
The form of the agent or composition of the present invention is not particularly limited, and the component (A) [and / or the component (B) described later] may be used as it is as an agent (for example, a powder) or a composition, and other components. It may be formulated together with (carrier, excipient, etc.).

Examples of other components include components generally used as raw materials for preparations, and are not particularly limited. For example, carriers, excipients, binders, disintegrants, lubricants, coating agents, colorants, and flavoring agents. Examples thereof include agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants and the like. These other components may be used alone or in combination of two or more.

The carrier is not particularly limited, and is, for example, animal and vegetable oils such as soybean oil, beef fat, and synthetic glyceride; hydrocarbons such as liquid paraffin, squalane, and solid paraffin; ester oils such as octyldodecyl myristate and isopropyl myristate; and cetostearyl. Higher alcohols such as alcohol and behenyl alcohol; Silicon resin; Silicone oil; Polyoxyethylene fatty acid ester, sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene block copolymer, etc. Surfactants; water-soluble polymers such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymers, polyethylene glycol, polyvinylpyrrolidone, methyl cellulose; lower alcohols such as ethanol, isopropanol; glycerin, propylene glycol, dipropylene glycol, sorbitol, etc. Polyhydric alcohols; sugars such as glucose and sucrose; inorganic powders such as silicic anhydride, aluminum magnesium silicate, and aluminum silicate; purified water and the like.

Examples of excipients include lactose, cornstarch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose, silicon dioxide and the like.

Examples of the binder include polyvinyl alcohol, gelatin, methyl cellulose, ethyl cellulose, gum arabic, tragant, gelatin, shelac, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, polyvinyl acetal diethylaminoacetate, corn starch and the like.

Examples of the disintegrant include corn starch, low-substituted hydroxypropyl cellulose, crospovidone, crystalline cellulose, precipitated calcium carbonate, croscarmellose sodium, calcium citrate, dextrin, pectin, calcium carboxymethyl cellulose and the like.

Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, light anhydrous silicic acid, sucrose fatty acid ester, and the like, and examples of the flavoring agent include cocoa powder, menthol, aromatic powder, peppermint oil, and dragon brain. Cinnamon powder is used.

The form (dosage form) of the agent or composition is not particularly limited, and for example, tablets, powders, fine granules, granules, dry syrups, coated tablets, orally disintegrating tablets, chewable tablets, capsules, soft capsules. , Syrups, oral solutions, troches, jellies, inhalants, suppositories, injections, ointments, eye drops, eye ointments, nasal drops, ear drops, poultices, lotions, topical solutions, sprays Examples thereof include agents, topical aerosols, creams, gels, tapes, buccal tablets, sublingual tablets, vaginal suppositories, vaginal tablets, and rectal soft capsules.

The form of administration (administration) of the agent or composition is not particularly limited, and may be oral administration or parenteral administration. Parenteral administration includes, for example, rectal administration, nasal administration, pulmonary administration, injection administration (for example, intravenous administration, intravertebral administration, intraepidural administration, intramuscular administration, subcutaneous administration, intraperitoneal administration). , Intra-arterial administration, intra-arterial administration, intracardiac administration, intracapsular administration, intradermal administration, intralesional administration, intraocular administration, intrathoracic administration, epidural administration, intrauterine administration, intraventricular administration) and the like.

In particular, since the component (A) [and / or the component (B) described later] often accompanies the proliferation of muscle cells and the increase in muscle mass as described above, the agent or composition of the present invention is a muscle. Oral administration may be preferably applied. On the other hand, oral administration may be preferably used from the viewpoint of simple administration.

In the agent or composition of the present invention, the amount of the component (A) [and / or the component (B) described later] is not particularly limited and can be appropriately selected depending on the dosage form, dosage and the like.

The dose (intake, dose) of the agent or composition of the present invention varies depending on the degree of disease / symptom, age, sex, body weight, administration form, type of salt, specific type of disease, and the like. For example, the amount of the component (A) per day may be about 0.4 to 40 mg / kg, preferably about 2 to 20 mg / kg, and more preferably about 4 to 8 mg / kg.

In particular, when Nikujuyo extract is used as a component containing the component (A), the dose of Nikujuyo extract is, for example, 1 to 6000 mg / kg, preferably 5 to 3000 mg / kg, and more preferably 10 to 1200 mg / kg. It may be about.

Administration may be divided into single doses or multiple doses.

The agent or composition of the present invention may be a drug or a pharmaceutical composition, or may be a quasi drug.

Moreover, the agent or composition of this invention may be in the form of food and drink. Therefore, the present invention also includes foods and drinks containing the component (A) [and / or the component (B) described later] (or the agent or composition). Further, from the viewpoint of the component (A) [and / or the component (B) described later] (or the agent or composition), the component (A) [and / or the component (B) described later] is a food or drink. It may be an additive.

In such foods and drinks, the preferred embodiment of the component (A) may be the same as described above.

Foods and drinks include general foods including so-called health foods, as well as health functional foods such as foods for specified health use and nutritional functional foods stipulated in the health functional food system of the Ministry of Health, Labor and Welfare, and supplements ( Dietary supplements), feeds, food additives and the like are also included in the foods and drinks of the present invention.

In addition, foods and drinks are intended for specific subjects [for example, for elderly people, patients or sick people (for example, neurological diseases, muscle diseases, sarcopenia, locomotive syndrome, paralysis, etc. (such as the above-exemplified diseases or symptoms)). (For patients who have), etc.] may be food and drink.

In order to use the component (A) in a food or drink, the food or drink can be produced as it is or mixed with various nutritional components and the like as raw materials for the food and drink such as processed meat and soft drinks.

When the ingredient (A) is used as a health food, dietary supplement, etc., for example, tablets, capsules (soft capsules, hard capsules, etc.), powders, granules, liquids (suspensions, syrups) are used by conventional means. Agents, etc.), emulsions, jellies, sticks, etc. can be prepared. The tablets also include disintegrating tablets (orally disintegrating tablets).

Foods and drinks may contain food additives (food additives). The food additive is not particularly limited, but for example, excipients (for example, wheat starch, corn starch, cellulose, lactose, sucrose, mannitol, sorbitol, xylitol, pregelatinized starch, casein, magnesium silicate aluminate, etc. Calcium silicate, etc.), excipients (eg, pregelatinized starch, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc.), disintegrants (eg, cellulose, hydroxypropylcellulose, corn starch, etc.), fluidizers (eg, light anhydrous silicic acid) , Sucrose fatty acid esters, etc.), oils (eg, soybean oil, sesame oil, olive oil, flaxseed oil, sesame oil, rapeseed oil, coconut oil, corn oil and other vegetable oils or animal / fish-derived oils), nutrients (eg, various Minerals, various vitamins, amino acids), fragrances, sweeteners, flavoring agents, coloring agents, solvents (ethanol), salts, surfactants, pH adjusters, buffers, antioxidants, stabilizers, gelling agents, increase Examples include adhesives, starches, encapsulants, suspending agents, coating agents, preservatives and the like.

The food additive may be used alone or in combination of two or more.

When the component (A) is used as a food and drink additive, the food and drink are not particularly limited, but for example, foods [for example, noodles (soba, udon, Chinese noodles, instant noodles, etc.), confectionery (candy, candy, etc.) Gum, chocolate, snacks (potato chips, etc.), biscuits, cookies, gummy, jelly, jam, butter, cream (cream puffs, etc.), cakes, breads, marine or processed livestock foods (kamaboko, ham, sausage, etc.), Dairy products (processed milk, fermented milk, etc.), fats and oils and processed fats and oils (salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressings, etc.), seasonings (sauce, sauce, etc.), retort foods (curry, stew) , Bowls, porridge, miscellaneous dishes, etc.), cold confectionery (ice cream, sorbet, shaved ice, etc.), fried foods (croquette, fried potatoes, fried chicken, etc.)], beverages (tea beverages, soft drinks, carbonated beverages, nutritional beverages, fruits Beverages, lactic acid beverages, etc.).

The blending amount of the component (A) in the above food and drink varies depending on the addition form and the administration form and can be selected from a wide range. For example, 0.0001% by weight or more (for example, 0.001 to 50% by weight). It may be 0.003% by weight or more (for example, 0.005 to 30% by weight), 0.01% by weight or more (for example, 0.05 to 10% by weight), and the like.

In the food and drink, the intake (or dose or dose) of the component (A) can be selected from the same range as described above.

<Method, other agent or composition>
As described above, the agent or composition (or component (A)) of the present invention can be applied to various uses. Therefore, the present invention also includes a method corresponding to the said use.

First, in the first method, the component (A) (or the agent or composition) causes the secretion of periostin and / or PKM2 (promotes the secretion of periostin and / or PKM2).

As described above, periostin and / or PKM2 proliferates muscle cells, increases muscles, and stretches or repairs axons. Therefore, the present invention also includes the following inventions.

(I) An agent or composition containing at least one component (B) selected from periostin and PKM2 for improving (treating) muscle cell proliferation, muscle gain (growth), and / or muscular atrophy. ..

(Ii) An agent or composition containing at least one component (B) selected from periostin and PKM2 for extending and / or repairing axons.

(Iii) Using at least one component (B) selected from periostin and PKM2 (or secreting component (B)), muscle cells proliferate, muscle gain (growth) and / or muscle atrophy How to improve (treat).

(Iv) A method of extending and / or repairing an axon using (or secreting) a component (B) of at least one selected from periostin and PKM2.

As described above, these agents, compositions and methods can be used for improving diseases and symptoms corresponding to them (for example, (1) to (6) below).

(1) Treatment and / or improvement of neurological disorders (2) Treatment and / or improvement of muscle diseases (3) Treatment and / or improvement of sarcopenia (4) Treatment and / or improvement of locomotive syndrome (5) Treatment and improvement of paralysis / Or improvement (6) Improvement (improvement) and / or maintenance of motor function and / or physical strength

In such agents, compositions and methods, periostin and PKM2 may be secreted in vivo or may be in vitro (such as those synthesized in vitro). .. For example, periostin and PKM2 may be recombinants or may be synthesized by an organic chemical method.
Further, in the above-mentioned agent, composition and method, at least the component (B) may be used, and the component (A) and the component (A) may be used in combination.

In the second method, component (A) (or the agent or composition) is used (eg, administered) to proliferate muscle cells, increase (grow) muscle and / or improve (treat) muscle atrophy. ). In this method, the axons may also be extended and / or repaired. Then, this method can be used, for example, for improving diseases and symptoms corresponding to these (for example, (1) to (6) above).

In the second method, muscle cells may be proliferated through (via) secretion of periostin and / or PKM2 to increase (increase) muscle and / or improve (treat) muscle atrophy. ..

In the above-mentioned agent, composition, method, etc., the mode of administration method, etc. may be the same as described above.
For example, the amount (dose, dose) of the component (B) may be 0.1 to 50 mg / kg, 0.5 to 10 mg / kg, 1 to 5 mg / kg, or the like. As a more specific dose of the component (B), 0.1 to 0.5 mg / kg, 1 to 5 mg / kg, 10 to 50 mg / kg and the like can be selected.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples, and many modifications are made in the art within the technical idea of the present invention. It is possible by a person with ordinary knowledge.

<Nikujuyo extract>
The Nikujuyo extract was extracted by adding 20 times the amount of water to Nikujuyo (Tochimoto Tenkaido, Osaka) and using a decoction machine (Uchida Wakanyaku, Tokyo). The water added to the crude drug was heated on high heat (10 to 15 minutes) until it boiled, and then heating on a low heat was continued until the total heating time reached 1 hour. After filtering the filtrate, it was filtered with a cotton swab when heated, and the filtrate was allowed to cool at room temperature and then frozen in liquid nitrogen. The powder dried in a freeze-dryer was used as an extract powder (yield 40.8%) and dissolved in physiological saline to obtain an extract of Nikujuyo.
In the Nikujuyo extract, the content of acteoside was 0.21% by weight, and the content of echinacoside was 0.35% by weight.

<Acteoside>
Acteoside (also known as verbascoside, kusaginin) was used by dissolving a commercially available product (Extrasynthesis, France) in physiological saline.

<Test animal>
(1) Spinal cord injury model mouse: A ddY mouse was obtained from Japan SLC (Hamamatsu, Japan). In this example, female and 8-week-old ddY mice were used.
(2) Mice for primary cultured skeletal muscle cells: ddY mice 3 days after birth were used.
(3) Mice for primary cultured neurons: 14-day-old embryonic ddY mice were used.
All mice were fed free feed and water and bred in a controlled environment at 22 ± 2 ° C., 50 ± 5% humidity, and a 12-hour light-dark cycle starting at 7 am.

<Primary culture of mouse skeletal muscle cells>
Three-day-old ddY mice were placed under anesthesia and the entire skeletal muscles of the left and right hind limbs were removed. Finely chopped muscle tissue was added to a degradation solution (1 mg / mL collagenase, 0.02 mg / mL dispase I, 2.5 mM calcium chloride in HBSS buffer) and incubated at 37 ° C. for 1 hour. Myocyte Growth Medium (Promo cell, Germany) was added to the sediment after centrifugation, and the cells were gently suspended by pipetting with a Pasteur pipette until the cell mass became almost invisible, and then 70 μm. Filtered with a nylon cell strainer (Falcon, USA). Cells were cultured on 8-well chamber slides (Falcon), white 96-well plates, and 6 cm culture dishes (Falcon).
To evaluate cell proliferation activity, muscle cells were seeded on a white 96-well plate at a density of 1,000 cells / well, and after 3 days of drug treatment, viable cells were measured with CellTiter-Glo reagent (Promega, USA). did.

<Primary culture of mouse cerebral cortex neurons>
The cerebral cortex of a 14-day-old ddY mouse fetus was cut into approximately 1 mm squares in a safety cabinet. After centrifuging at 700 rpm for 3 minutes, the supernatant was removed, and 2 mL of 0.05% trypsin-0.53 mM EDTA solution (Life Technologies) was added to the precipitate and suspended. Incubate at 37 ° C. for 15 minutes with stirring every 5 minutes, add 4 mL of medium, centrifuge at 700 rpm for 3 minutes to remove the supernatant, and deposit 600 U / mL DNase I (Life Technologies) -0.03% trypsin. 2 mL of Inhibitor (Life Technologies) -PBS solution was added and suspended. Further, the mixture was incubated at 37 ° C. for 15 minutes with stirring every 5 minutes, 4 mL of the medium was added, and the medium was centrifuged at 700 rpm for 3 minutes. 4 mL of calcium / magnesium-free HBSS (Hank's balanced salt solution) was added to the sediment and centrifuged at 700 rpm for 3 minutes. 4 mL of medium was added to the sediment. The cells were gently suspended in a pasteur pipette with a roasted tip until the cell mass disappeared, and then filtered through a 70 μm nylon cell strainer (Falcon). Cell culture was performed on 8-well chamber slides (Falcon). The day before, the coating [Poly-D-lysine (PDL, Sigma-Aldrich) was diluted with PBS to 0.005 mg / mL, sprinkled, and incubated at 37 ° C. Wash twice with sterile purified water on the day of culture. ], The cells were seeded. Culturing was started at 37 ° C., 10% CO ₂ , saturated steam, and after 5 hours, the whole medium was replaced with a new medium containing B-27 supplement (Invitrogen) instead of horse serum. After conditioning medium or drug treatment, immunostaining was performed in the evaluation of axonal extension.

For immunostaining, after the end of the drug treatment period, the medium was removed, the cells were washed with PBS, 4% paraformaldehyde (PFA, Wako Pure Drug) -PBS solution was added, and the cells were allowed to stand at room temperature for 2 hours to fix the cells. Washing was performed twice for 5 minutes with 0.3% triton X-100 (Wako Pure Drug) -PBS solution. Then, the primary antibody solution [0.3% Triton X-100-PBS solution, 1% normal goat serum (NGS, Wako Pure Drug), primary antibody] was added, and the mixture was reacted overnight at 4 ° C. It was.

As the primary antibody, mouse anti-phosphorylated NF-H monoclonal antibody (1: 250) (COVANCE, Emeryville, CA, USA) and rabbit anti-MAP2 polyclonal antibody (1: 2000) (abcam, Cambridge, UK) were used. There was. The next day, the primary antibody solution was removed, and after washing twice with 0.3% Triton X-100-PBS solution for 5 minutes, the secondary antibody solution [0.3% Triton X-100-PBS solution, Alexa Fluor 594-labeled goat anti-mouse IgG antibody (1: 200) (Molecular Probes, Eugene, OR, USA), Alexa Fluor 488-labeled goat anti-rabbit IgG antibody (1: 200) (Molecular Probes, Eugene, OR, USA)] In addition, the reaction was carried out at room temperature under shading for 2 hours. After the reaction, the secondary antibody solution was removed, the cells were washed twice with PBS for 5 minutes, the nuclei were stained with DAPI staining, and then encapsulated with Aqua Poly Mount (Polysciences, Warrington, USA).

Images were acquired from wells treated with each drug using a fluorescence microscope (Cell Observer, Carl Zeiss, Germany) with a 10-20x objective lens. For those images, the length of pNF-H-positive axons on the entire screen and the MAP2-positive nerve cells in each image were measured using the image analysis software MetaMorph (Molecular Device, Tokyo), and per nerve cell. The length of the axon was calculated.

<Spinal Cord Injury (SCI) Mouse: Contusion Model>
8-10 week old female ddY mice were bruised. That is, the 8th to 9th lumbar vertebrae of the mouse were excised according to a conventional method, and a weight of 3 cm to 6.5 g in height was once dropped onto the 11th to 12th thoracic spines using a stereotaxic fixation device (manufactured by Narishige) to cause contusion. Gave. After that, surgical procedures such as suturing were performed according to a conventional method. Hereinafter, this mouse is referred to as an SCI crush model. Mice were maintained under constant environmental conditions (22 ± 2 ° C., 50 ± 5% humidity, 12-hour light-dark cycle starting at 7:00 am) in a state where food and water could be freely ingested.

<Spinal cord injury (SCI) mouse: complete amputation model>
The 10th lumbar vertebrae of 8-week-old female ddY mice were excised to expose the 1st lumbar spine and completely vertically cut with Micro Caesars. After that, surgical procedures such as suturing were performed according to a conventional method. Hereinafter, this mouse is referred to as a SCI complete cleavage model. Mice were maintained under constant environmental conditions (22 ± 2 ° C., 50 ± 5% humidity, 12-hour light-dark cycle starting at 7:00 am) in a state where food and water could be freely ingested.

<Evaluation of hind limb motor function by TMS>
Mice after administration of the extract and the like were individually moved to an open field (42 cm × 48 cm × 15 cm) and observed for 5 minutes to evaluate hindlimb motor function. Basso Mouse Scale (BMS, Engesser-Cesar C, Anderson AJ, Basso DM et al. (2005).), Which is commonly used as a criterion for evaluating the motor function of the hind limbs in a model test of spinal cord injury. In order to improve the accuracy of the test, we evaluated the movement behavior in the open field using the Toyama Mouse Scale (TMS) of 0 to 30 points, which was modified from BMS.

<Statistical processing>
In this example, the results obtained were subjected to the following statistical processing:
One-way ANOVA, post-Dunnett test, and corresponding t-test are available at Graphpad Prism 6 (Graphpad Software, La Jolla, La Jolla, Inc.) California, USA). ^* P <0.05, ^** P <0.01, ^*** P <0.001 are statistically significant, and the average value is shown together with the standard error.

1. 1. Axon extension action (1-1) The culture supernatant (conditioned medium) obtained by drug-treating primary cultured mouse skeletal muscle cells was treated with primary cultured mouse cerebral cortex neurons, and the axon extension action was examined. The results are shown in FIG. The white column in the figure is a conditioned medium obtained by treating skeletal muscle cells with a solvent (sterilized water) and treating nerve cells.

A in FIG. 1 shows the results of treating skeletal muscle cells with Nikujuyo extract (final concentration in medium: 10 μg / mL, 100 μg / mL), and the conditioned medium significantly axons in the Nikujuyo extract treatment group. It can be seen that the density has increased.

In addition, B in FIG. 1 shows the results of treating skeletal muscle cells with acteosides (final concentrations in the medium: 1 nM, 100 nM, 1000 nM), and it can be seen that the axon density was significantly increased by the conditioned medium.

(1-2) Fig. 2 shows the results of directly treating primary cultured mouse cerebral cortical neurons with a drug and examining the axon extension action. The white column in the figure was treated by adding a solvent (sterilized water) to the medium instead of the drug.

FIG. 2A shows the results of treatment of nerve cells with Nikujuyo extract (final concentration in medium: 10 μg / mL), and an increasing tendency of axon extension was observed.

FIG. 2B shows the results of treatment of nerve cells with acteoside (final concentration in medium: 1 μM), and a significant axon extension effect was observed.

2. Proliferation of muscle cells FIG. 3 shows the results of treating the skeletal muscle cells of the primary cultured mouse with a drug and examining the proliferative effect of the muscle cells.

As is clear from the results of FIG. 3, the skeletal skeletal structure was treated with Nikujuyo extract (final concentration in medium: 1,100,100 μg / mL) and acteoside (final concentration in medium: 1,100,1000 nM). The proliferation of muscle cells was significantly promoted. IGF-1 was used as a positive control.

3. 3. Improvement of motor function in the chronic phase decreased due to spinal cord injury (3-1) After 45 days have passed since the mouse was injured by spinal cord injury and the so-called post-injury chronic phase was reached, the biceps femoris extract was applied to the hind limb biceps femoris. Was intramuscularly injected (2.0 mg / limb, intramuscular injection into the left and right hind limbs, 3 times / week), and the transition of hind limb motor function during the drug administration period over 35 days was examined. The results are shown in FIG.

FIG. 4A shows the results of evaluation on the Basso Mouse Scale (BMS), and showed a tendency for the motor function to improve in the Nikujuyo extract injection group.

FIG. 4B shows the results of evaluation on the Toyama Mouse Scale (TMS), and the Nikujuyo extract-administered group showed a significant improvement in hindlimb motor function.

(3-2) Mice were injured by spinal cord injury due to crush, and after 45 days had passed, after the so-called chronic phase after injury, acteoside was intramuscularly injected into the biceps femoris of the hind limbs (0.1 mg / limb, to the left and right hind limbs). The transition of hindlimb motor function during the drug administration period of 35 days with intramuscular injection (3 times / week) was examined. The results are shown in FIG.

FIG. 5A shows the results evaluated by BMS and FIG. 5B shows the results evaluated by TMS, and in both cases, a significant hindlimb motor function improving effect was observed in the acteoside injection group.

(3-3) Mice were injured by complete spinal cord transection, and 35 days later, after the so-called chronic phase after injury, Nikujuyo extract was intramuscularly injected into the biceps femoris of the hind limbs (2.0 mg / limb, left and right). Intramuscular injection into the hind limbs (3 times / week), the transition of hind limb motor function during the drug administration period over 56 days was examined. The results are shown in FIG.

FIG. 6A shows the results evaluated by BMS and FIG. 6B shows the results evaluated by TMS, and in both cases, a significant hindlimb motor function improving effect was observed in the Nikujuyo extract injection group.

4. Increased muscle weight Mice were injured by complete spinal cord transection, and 35 days later, after the so-called chronic phase after injury, Nikujuyo extract was intramuscularly injected into the biceps femoris of the hind limbs (2.0 mg / limb, left and right hind limbs). Wet weight of the gastrocnemius muscle was examined after intramuscular injection, 3 times / week) and drug administration for 56 days. The results are shown in FIG.

As is clear from the results of FIG. 7, the skeletal muscle weight was significantly reduced in the saline-administered mice in the amputated group as compared with the uncut group. However, an increase in gastrocnemius muscle weight was observed by injection of Nikujuyo extract.

5. Oral administration (5-1) Mice are injured by spinal cord injury due to crushing, and after 51 days, the so-called post-injury chronic phase is followed by oral administration of Nikujuyo extract, and the hind limbs from the time of spinal cord injury to the end of drug administration. The transition of motor function was investigated. The dose was 500 mg / kg / day, and administration was performed for 138 days. The results are shown in FIG.

FIG. 8A is the result of evaluation by BMS, and FIG. 8B is the result of evaluation by TMS. In all cases, a significant hindlimb motor function improving effect was observed in the oral administration group of Nikujuyo extract.

(5-2) Spinal cord injury due to crushing was given to the mouse, and 51 days later, after the so-called chronic phase after the injury, the biceps femoris extract was orally administered by gastrocnemius, and the tibialis anterior muscle after the drug administration was completed. The wet weight of the biceps femoris, tibialis anterior, vastus lateralis, and gastrocnemius muscles) was examined, and the ratio to body weight (muscle wet weight / body weight) was calculated. The dose was 500 mg / kg / day, and the dose was 138 days.
The results are shown in FIG. In FIG. 9, Sham indicates the sham surgery group, SCI / Veh indicates the solvent administration to the spinal cord injury group, and SCI / NJ indicates the Nikujuyo extract administration to the spinal cord injury group. The solvent was sterilized water, and a solution of Nikujuyo extract in sterilized water in an amount of 5% by mass was administered.

As is clear from the results of FIG. 9, a significant increase in muscle weight was observed in the gastrocnemius muscle and the tibialis anterior muscle. In addition, the vastus lateralis muscle and the biceps femoris also tended to increase muscle weight due to the administration of Nikujuyo extract.

6. We identified proteins that increase to the conditioned medium obtained by acteoside treatment of skeletal muscle cells of primary cultured mice that promote the secretion of periostin and PKM2 . The results are shown in FIG.

FIG. 10A shows the results of electrophoresing and silver staining of a conditioned medium obtained by treating skeletal muscle cells with acteoside. Two bands increased in a dose-dependent manner with acteoside.
Each of these bands was excised together with the gel, and after in-gel trypsin digestion, nano LC-MS / MS analysis was performed. The results of the mass pattern of the peptide fragments indicated that the two bands could be periostin and PKM2.

FIG. 10B shows the results of Western blotting of the conditioned medium obtained by treating skeletal muscle cells with acteoside with an antibody of periostin. FIG. 10C shows the result of dissolving acteoside in sterile water and adding it to the medium to treat skeletal muscle cells. This is the result of Western blotting of the conditioned medium with an antibody of PKM2. It was confirmed that periostin and PKM2 were certainly increased in the conditioned medium obtained by treating skeletal muscle cells with acteoside.

7. Function by periostin / PKM2 (7-1) Primary cultured mouse Cerebral cortex neurons were treated with recombinant periostin or recombinant PKM2 (both commercially available), and the axon extension effect was examined. The results are shown in FIG.

FIG. 11A shows the results of treating nerve cells by dissolving recombinant periostin in sterile water and adding it to a medium, and a significant axon extension effect was observed.

FIG. 11B shows the results of treating the nerve cells by dissolving the recombinant PKM2 in sterile water and adding it to the medium, and a significant axon stretching effect was observed at a dose of 10,100,1000 ng / ml.

(7-2) Primary cultured mouse skeletal muscle cells were treated with recombinant periostin or recombinant PKM2 (both commercially available), and the cell proliferation effect was examined. The results are shown in FIG.

FIG. 12A shows the results of treating skeletal muscle cells by dissolving recombinant periostin in sterile water and adding it to the medium, and the proliferation was significantly promoted.

FIG. 12B shows the results of treating the skeletal muscle cells by dissolving the recombinant PKM2 in sterile water and adding it to the medium, and the proliferation was significantly promoted.

8. Chronic spinal cord injury due to acteoside Improvement of motor function, improvement of muscle atrophy and increase of presynaptic projection to motor nerves (8-1) Spinal cord injury due to crushing in mice, 30 days later, so-called post-injury chronic phase After that, acteoside was injected intramuscularly (0.2 mg / limb, intramuscular injection into the left and right hind limbs, 3 times / week), and the transition of hind limb motor function from the injury of SCI to the end of drug administration was investigated. The administration period was 62 days. The results are shown in FIG.

FIG. 13A is the result of evaluation by BMS, and FIG. 13B is the result of evaluation by TMS. In all cases, a significant hindlimb motor function improving effect was observed in the acteoside intramuscular injection group.

(8-2) Mice were injured by spinal cord injury due to crush, and 30 days later, after the so-called chronic phase after injury, acteoside was intramuscularly injected (0.2 mg / limb, intramuscular injection into the left and right hind limbs, 3 times / Week), the wet weight of the hindlimb skeletal muscles (biceps femoris, tibialis anterior muscle, vastus lateralis muscle, gastrocnemius muscle) after the end of drug administration was examined, and the ratio to the body weight (muscular wet weight / body weight) was calculated. The administration period was 62 days.
The results are shown in FIG. In FIG. 14, Sham indicates sham surgery group, SCI / Veh indicates solvent administration to the spinal cord injury group, and SCI / AC indicates acteoside administration to the spinal cord injury group. The solvent was physiological saline.

As is clear from the results of FIG. 14, a significant increase in muscle weight was observed in the biceps femoris and tibialis anterior muscles. In addition, the vastus lateralis muscle and gastrocnemius muscle also tended to increase muscle weight due to the administration of acteoside.

(8-3) Mice were injured by spinal cord injury due to crush, and after 30 days, acteoside was intramuscularly injected (0.2 mg / limb, intramuscular injection into left and right hind limbs, 3 times /) in the so-called chronic phase after injury. Week), the lumbar spinal cord was removed from the spinal cord tissue after drug administration, where the motor nerves projecting to the biceps femoris, tibialis anterior, vastus lateralis, and gastrocnemius muscles were localized. A 14 μm-thick continuous sagittal section was prepared and immunostained with an antibody against the motor nerve marker choline acetyltransferase and an antibody against the presynaptic marker synaptophysin. The area of presynapses projected onto motor neurons was quantified. The results are shown in FIG. In FIG. 15, Sham indicates sham surgery group, SCI / Veh indicates solvent administration to the spinal cord injury group, and SCI / AC indicates acteoside administration to the spinal cord injury group. The solvent was physiological saline.

As is clear from the results of FIG. 15, administration of acteoside increased presynapses that project to motor neurons.

9. Improvement of motor function in muscularly atrophied mice by Nikujuyo extract (9-1) A cast was attached to fix the joints of both hind limbs of the mouse in the extended position, and the cast was removed 12 days later. This is a mouse model of sarcopenia in which significant skeletal muscle atrophy is observed in the cast-attached group compared to the non-cast-attached group (sham-operated group). During the cast wearing period, Nikujuyo extract was orally administered daily. The dose was 50 mg / kg / day and 100 mg / kg / day. The day after the cast was removed, the motor function was evaluated by a beam test. The results (results of motor function on the 13th day when the Nikujuyo extract was dissolved in sterile water and orally administered to muscular atrophied mice) are shown in FIG. The beam test observes the mouse walking on bars with widths of 5 mm, 9 mm, and 12 mm, scores the frequency with which the left and right hind limbs slide down, and calculates the total score.The higher the score, the more you can walk without failure. Shown.

As is clear from the results of FIG. 16, the Nikujuyo extract had a significant improving effect on the motor function of the muscular atrophied mice.

INDUSTRIAL APPLICABILITY The present invention can provide an agent or composition useful for muscle cell proliferation, muscle gain, and secretion of periostin and PKM2.

Claims (13)

An agent or composition containing at least one component (A) selected from acteosides, echinacosides and salts thereof, for promoting the secretion of periostin and / or pyruvate kinase M2. An agent or composition containing at least one component (A) selected from acteosides, echinacosides and salts thereof, for improving muscle cell proliferation, muscle gain and / or muscle atrophy. The agent or composition according to claim 2, further for extending and / or repairing an axon. An agent containing at least one component (B) selected from periostin and PKM2 for axon extension, axon repair, improvement of myocyte proliferation, muscle gain, and / or improvement of muscle atrophy. Or the composition. The agent or composition according to claim 4, wherein the component (B) is present in vitro. The agent or composition according to any one of claims 1 to 5, for use in at least one application selected from the following (1) to (6).
(1) Treatment and / or improvement of neurological disorders (2) Treatment and / or improvement of muscle disorders (3) Treatment and / or improvement of sarcopenia (4) Treatment and / or improvement of locomotive syndrome (5) Treatment and / or improvement of paralysis / Or improvement (6) Improvement and / or maintenance of motor function and / or physical strength The agent or composition according to claim 6, wherein neurological disease, muscle disease, sarcopenia, locomotive syndrome and paralysis are symptoms of the chronic phase. The agent or composition according to any one of claims 1 to 7, for treating and / or ameliorating spinal cord injury in the chronic phase. The agent or composition according to any one of claims 1 to 3 and 6 to 8, which contains at least an acteoside as the component (A). The agent or composition according to any one of claims 1 to 3 and 6 to 9, which contains a sardine component as a component containing the component (A). The agent or composition according to any one of claims 1 to 10, which is for intramuscular injection or oral use. The agent or composition according to any one of claims 1 to 11, which is a food or drink. The agent or composition according to any one of claims 1 to 12, which is a supplement, a health food, a food with a functional claim, a food with a nutritional function, or a food for specified health use.