JP2016135765A - Prophylactic agent for muscular contracture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical material or a food material which contains a carotene-oxidized derivative derived from a land animal, a land plant, or a microorganism, as an active ingredient and can ameliorate muscle tonus, can alleviate muscular fatigue, or can prevent muscular contracture.SOLUTION: The invention provides a muscle tonus ameliorating agent, a muscle fatigue alleviating agent, and a muscle contracture preventive agent that contain, as an active ingredient, at least one or more kinds of lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin that are carotene-oxidized derivatives containing at least one of a hydroxyl group, a carbonyl group, and an ether group.SELECTED DRAWING: Figure 2

Description

  The present invention relates to an agent for improving muscle tone, an agent for reducing muscle fatigue, and an agent for preventing muscle contracture, which contain terrestrial animals, terrestrial plants or microorganism-derived carotene oxidation derivatives as active ingredients.

In a modern society with irregular life and stress, it is recommended that appropriate exercise and sports be performed to maintain health and prevent stress, including prevention of lifestyle-related diseases. However, if a person who does not usually move their body suddenly exercises or plays a sport, muscle fatigue may occur immediately, or severe muscle pain or muscle fatigue may occur after exercise. May be the reason why you cannot continue exercising on a daily basis.
Moreover, in elderly people and long-term caregivers, moderate exercise or rehabilitation is used as a means to prevent athletic ability decline, but there is also the difficulty of maintaining motivation and the risk of injury, In reality, it is quite difficult and a more effective method is desired. Therefore, there is a demand for an effective method for muscular pain and muscular fatigue, such as improving persistence while preventing muscular fatigue, or reducing severe muscular pain and muscular fatigue after exercise. In addition, stiff muscles (back pain, narrow joint range of motion, stiff shoulders, etc.) that occur in elderly people, long-term care patients, etc. who are unable to move their bodies, are regarded as a problem that causes a reduction in exercise capacity. There is a further need for means to assist or replace exercise, rehabilitation and the like.

  As one means for solving these problems, it is conceivable to ingest daily food components having an effect of improving motor functions such as endurance and muscle strength. Examples thereof include hawthorn extract (Patent Document 1), coenzyme Q10, carnitine compound (Patent Document 2), glutamine peptide (Patent Document 3), and the like.

  On the other hand, carotenoid is a general term for the hydrocarbons carotenes in which eight isoprenoid units are bonded. Carotenoids are natural pigments that are distributed in plants, microorganisms and animals, have a physiological action as provitamin A, such as β-carotenes, and active ingredients of herbal medicines such as crocin and crocetin, and coloring of foods containing these Some important fees are included. In plants and photosynthetic bacteria, it is known as an auxiliary enzyme in light energy transmission (Non-patent Document 1). Recently, many new actions have been found in carotenoids, so-called marine carotenoids contained in marine organisms such as seafood and seaweed.

  For example, fucoxanthin, which is one of fat-soluble substances contained in seaweeds such as hijiki and seaweed, has anti-obesity action (Non-patent Document 2), neutral fat absorption inhibitory action (Patent Document 4), and the like.

  In addition, for example, astaxanthin contained in crustaceans such as shrimp and crab and used as a red pigment has an in vivo antioxidant effect (Patent Document 5), and an liver function improving effect (patent) Document 6) has been found. Furthermore, astaxanthin has been found to have an effect of improving muscle damage and disease (Patent Document 7) and to have an anti-fatigue effect (Non-Patent Document 3 and Non-Patent Document 4).

  However, when marine carotenoids are applied to foods and drinks such as dairy products, for example, odors derived from raw materials become a problem, and problems remain in terms of flavor. Therefore, a material that can be easily extracted and purified from land animals and plants and microorganisms, not marine organisms, and has an effect on muscle fatigue has been demanded.

JP-A-8-47381 JP-A-2005-97161 JP-A-2005-97162 Special table 2001-514215 gazette JP-A-2-49091 JP-A-9-124470 Special table 2001-514215 gazette

Natural Product Chemistry Rev. 4th page, 145 pages, Nankodo H. Maeda et al., Biochemical and Biophysical Research Communication, 332 (2005) 392-397. Kazuyoshi Yazawa, FOOD Style 21, 9 (10), pp20-23 (2005) Nagata, et al., Science of Fatigue and Rest, 18 (1), pp35-45 (2003)

  The present invention has been made in view of the above situation, and the problem to be solved by the present invention is to improve muscle tone suitable for food and drink and pharmaceutical applications from land-based animals and plants and microorganism-derived carotenoids, It is to provide food materials and pharmaceutical materials that can reduce fatigue or prevent muscle contracture, and at the same time, improve muscle tone using the above materials, reduce muscle fatigue or prevent muscle contracture It is to provide a food composition and a pharmaceutical composition.

  In view of the above-described conventional problems, the present inventors have conducted extensive research, and as a result, terrestrial animals, land plants, or microorganism-derived carotene-oxidized derivatives improve muscle tone, reduce muscle fatigue, or prevent muscle contractures. It was found to have an action.

That is, the present invention relates to the following [1] to [72].
[1] A muscle tone improving agent comprising a caroten-oxidized derivative derived from land animals, land plants or microorganisms as an active ingredient.
[2] The muscle tone improving agent according to [1], wherein the terrestrial animal, land plant or microorganism-derived carotene oxidation derivative contains at least one of a hydroxyl group, a carbonyl group, and an ether group.
[3] The above terrestrial animal, terrestrial plant or microorganism-derived carotene oxidation derivative is at least lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin The muscle tone improving agent according to [1] or [2], wherein one or more active ingredients are used.
[4] The muscle tone improving agent according to any one of [1] to [3], wherein an effective amount of the muscle tone improving agent is 0.1 to 1500 mg / 60 kg body weight.
[5] A pharmaceutical composition effective for improving muscle tone, comprising 0.01 to 95% by mass of the muscle tone improving agent according to any one of [1] to [4].
[6] A food and drink effective for improving muscle tone, comprising 0.0001 to 10% by mass of the muscle tone improving agent according to any one of [1] to [4].
[7] A muscle fatigue reducing agent comprising a carotene oxidation derivative derived from a land animal, a land plant or a microorganism as an active ingredient.
[8] The muscle fatigue-reducing agent according to [7], wherein the terrestrial animal, land plant, or microorganism-derived carotene oxidation derivative contains at least one of a hydroxyl group, a carbonyl group, and an ether group.
[9] The terrestrial animal, terrestrial plant or microorganism-derived carotene oxidation derivative is at least lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin The muscle fatigue-reducing agent according to [7] or [8], comprising one or more active ingredients.
[10] The muscle fatigue reducing agent according to any one of [7] to [9], wherein an effective amount of the muscle fatigue reducing agent is 0.1 to 1500 mg / 60 kg body weight.
[11] A pharmaceutical composition effective for reducing muscle fatigue, comprising 0.01 to 95% by mass of the muscle fatigue reducing agent according to any one of [7] to [10].
[12] A food and drink effective for reducing muscle fatigue, comprising 0.0001 to 10% by mass of the muscle fatigue reducing agent according to any one of [7] to [10].
[13] A muscular contracture preventive agent comprising a land animal, a land plant, or a microorganism-derived carotene-oxidized derivative as an active ingredient.
[14] The muscle contracture preventive agent according to [13], wherein the terrestrial animal, land plant, or microorganism-derived carotene oxidation derivative contains at least one of a hydroxyl group, a carbonyl group, and an ether group.
[15] The carotenylated derivative derived from land animals, land plants or microorganisms is at least lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin The agent for preventing muscle contracture according to [13] or [14], which comprises one or more active ingredients.
[16] The agent for preventing muscle contracture according to any one of [13] to [15], wherein an effective amount of the agent for preventing muscle contracture is 0.1 to 1500 mg / 60 kg body weight.
[17] A pharmaceutical composition effective for preventing muscle contracture, comprising 0.01 to 95% by mass of the agent for preventing muscle contracture according to any one of [13] to [16].
[18] A food and drink effective for preventing muscle contracture, comprising 0.0001 to 10% by mass of the muscle contracture preventing agent according to any one of [13] to [16].
[19] A method for improving muscle tone, comprising a step of administering to a subject a pharmaceutically effective amount of a carotenylated derivative derived from a land animal, a land plant or a microorganism.
[20] The method according to [19], wherein the caroten oxidation derivative derived from land animals, land plants, or microorganisms includes at least one of a hydroxyl group, a carbonyl group, and an ether group.
[21] The terrestrial animal, terrestrial plant or microorganism-derived carotene oxidation derivative is at least lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin The method according to [19] or [20], wherein one or more active ingredients are used.
[22] The method according to any one of [19] to [21], wherein the effective amount is 0.1 to 1500 mg / 60 kg body weight.
[23] A pharmaceutical composition effective for improving muscle tone, comprising a step of blending 0.01 to 95% by mass of the muscle tone improving agent according to any one of [1] to [4] How to do.
[24] A food or drink effective for improving muscle tone is produced, comprising a step of adding 0.0001 to 10% by mass of the muscle tone improving agent according to any one of [1] to [4]. Way for.
[25] A method for reducing muscle fatigue, comprising a step of administering to a subject a pharmaceutically effective amount of a carotenylated derivative derived from a land animal, a land plant or a microorganism.
[26] The method according to [25], wherein the carotene oxidation derivative derived from a land animal, a land plant, or a microorganism contains at least one of a hydroxyl group, a carbonyl group, and an ether group.
[27] The carotenylated derivative derived from land animals, land plants or microorganisms is at least lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin The method according to [25] or [26], wherein one or more active ingredients are used.
[28] The method according to any one of [25] to [27], wherein the effective amount is 0.1 to 1500 mg / 60 kg body weight.
[29] To produce a pharmaceutical composition effective for reducing muscle fatigue, comprising the step of blending 0.01 to 95% by mass of the muscle fatigue reducing agent according to any one of [7] to [10]. the method of.
[30] For producing a food or drink effective for reducing muscle fatigue, comprising adding 0.0001 to 10% by mass of the muscle fatigue reducing agent according to any one of [7] to [10]. Method.
[31] A method for preventing muscle contracture, comprising a step of administering to a subject a pharmaceutically effective amount of a carotenylated derivative derived from a land animal, a land plant or a microorganism.
[32] The method according to [31], wherein the carotene oxidation derivative derived from land animals, land plants, or microorganisms includes at least one of a hydroxyl group, a carbonyl group, and an ether group.
[33] The above terrestrial animal, terrestrial plant or microorganism-derived carotene oxidation derivative is at least lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin The method according to [31] or [32], wherein one or more active ingredients are used.
[34] The method according to any one of [31] to [33], wherein the effective amount is 0.1 to 1500 mg / 60 kg body weight.
[35] A pharmaceutical composition effective for the prevention of muscle contracture, comprising the step of blending 0.01 to 95% by mass of the agent for preventing muscle contracture according to any one of [13] to [16] How to do.
[36] A food or drink effective for preventing muscle contracture is produced, comprising a step of adding 0.0001 to 10% by mass of the muscle contracture preventing agent according to any one of [13] to [16]. Way for.
[37] Use of a carotenylated derivative derived from a land animal, a land plant, or a microorganism in the production of a muscle tone improving agent.
[38] The use according to [37], wherein the carotene oxidation derivative derived from a land animal, a land plant, or a microorganism contains at least one of a hydroxyl group, a carbonyl group, and an ether group.
[39] The terrestrial animal, terrestrial plant or microorganism-derived carotene oxidation derivative is at least lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin Use as described in [37] or [38] which uses 1 or more types as an active ingredient.
[40] The use according to any one of [37] to [39], wherein the effective amount of the muscle tone improving agent is 0.1 to 1500 mg / 60 kg body weight.
[41] Use of the muscle tone improving agent according to any one of [1] to [4] in the production of a pharmaceutical composition effective for improving muscle tone.
[42] Use of the muscle tone improving agent according to any one of [1] to [4] in the production of a food or drink effective for improving the muscle tone.
[43] Use of a carotene-oxidized derivative derived from a land animal, a land plant or a microorganism in the manufacture of a muscle fatigue reducing agent.
[44] The use according to [43], wherein the carotene oxidation derivative derived from a land animal, a land plant, or a microorganism contains at least one of a hydroxyl group, a carbonyl group, and an ether group.
[45] The above terrestrial animal, terrestrial plant or microorganism-derived carotene oxidation derivative is at least lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin Use as described in [43] or [44] which uses 1 or more types as an active ingredient.
[46] The use according to any one of [43] to [45], wherein the effective amount of the muscle fatigue reducing agent is 0.1 to 1500 mg / 60 kg body weight.
[47] Use of the muscle fatigue reducing agent according to any one of [7] to [10] in the production of a pharmaceutical composition effective for reducing muscle fatigue.
[48] Use of the muscle fatigue reducing agent according to any one of [7] to [10] in the production of a food and drink effective for reducing muscle fatigue.
[49] Use of a carotene-oxidized derivative derived from a land animal, a land plant or a microorganism in the manufacture of a muscle contracture preventive agent.
[50] The use according to [49], wherein the carotene oxidation derivative derived from a land animal, a land plant, or a microorganism contains at least one of a hydroxyl group, a carbonyl group, and an ether group.
[51] The terrestrial animal, terrestrial plant or microorganism-derived carotene oxidation derivative is at least lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin Use as described in [49] or [50] which uses 1 or more types as an active ingredient.
[52] The use according to any one of [49] to [51], wherein an effective amount of the agent for preventing muscle contracture is 0.1 to 1500 mg / 60 kg body weight.
[53] Use of the agent for preventing muscle contracture according to any one of [13] to [16] in the production of a pharmaceutical composition effective for preventing muscle contracture.
[54] Use of the muscle contracture preventive agent according to any one of [13] to [16] in the production of a food or drink effective for preventing muscle contracture.
[55] A carotene-oxidized derivative derived from land animals, land plants, or microorganisms for use in improving muscle tone.
[56] The carotene oxidized derivative according to [55], which contains at least one of a hydroxyl group, a carbonyl group, and an ether group.
[57] At least one or more of lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin, [55] or [56] The carotene-oxidized derivative according to 1.
[58] The carotene-oxidized derivative according to any one of [55] to [57], wherein an effective amount for use in improving muscle tone is 0.1 to 1500 mg / 60 kg body weight.
[59] The muscle tone improving agent according to any one of [1] to [4], which is used for producing a pharmaceutical composition effective for improving muscle tone.
[60] The muscle tone improving agent according to any one of [1] to [4] for use in the production of a food or drink effective for improving the muscle tone.
[61] A carotene-oxidized derivative derived from land animals, land plants, or microorganisms for use in reducing muscle fatigue.
[62] The carotene oxidized derivative according to [61], which contains at least one of a hydroxyl group, a carbonyl group, and an ether group.
[63] The active ingredient is at least one of lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin, [61] or [62] The carotene-oxidized derivative according to 1.
[64] The carotene-oxidized derivative according to any one of [61] to [63], wherein an effective amount for use in reducing muscle fatigue is 0.1 to 1500 mg / 60 kg body weight.
[65] The muscle fatigue reducing agent according to any one of [7] to [10] for use in the production of a pharmaceutical composition effective for reducing muscle fatigue.
[66] The muscle fatigue reducing agent according to any one of [7] to [10] for use in the production of a food or drink effective for reducing muscle fatigue.
[67] A carotene-oxidized derivative derived from land animals, land plants, or microorganisms for use in preventing muscle contractures.
[68] The carotene-oxidized derivative according to [67], which contains at least one of a hydroxyl group, a carbonyl group, and an ether group.
[69] At least one or more of lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin, [67] or [68] The carotene-oxidized derivative according to 1.
[70] The carotene-oxidized derivative according to any one of [67] to [69], wherein an effective amount for use in preventing muscle contracture is 0.1 to 1500 mg / 60 kg body weight.
[71] The agent for preventing muscle contracture according to any one of [13] to [16], which is used for producing a pharmaceutical composition effective for preventing muscle contracture.
[72] The agent for preventing muscle contracture according to any one of [13] to [16], which is used for producing a food or drink effective for preventing muscle contracture.

  The muscle tone improving agent, muscle fatigue reducing agent or muscle contracture preventing agent of the present invention improves the muscle tone produced by exercise and daily movement, reduces muscle fatigue, or prevents muscle contracture. Useful for food and medicine. The muscle tone improving agent, muscle fatigue reducing agent or muscle contracture preventing agent of the present invention comprises a land animal, a land plant, or a microorganism-derived carotene-oxidized derivative as an active ingredient, and has a rich dietary experience. In addition to low side effects and high safety, the odor derived from raw materials is far less than carotenated derivatives derived from marine organisms such as conventional seafood and seaweed, and is superior in flavor.

An example of muscle contraction measured in the example is shown. The vertical axis represents muscle tension (g), the horizontal axis represents time, and contraction every other minute was observed. The maximum muscle tension and the minimum muscle tension (muscle tonus) appear as the upper and lower ends of each vertical bar. The contraction force (g) and muscle tone (g) can be calculated from this chart, and a muscle tone measurement chart can be created. 2 is a muscle tone measurement chart of rat soleus muscle to which lutein or olive oil of Example 1 was orally administered. Expressed as mean ± standard error. At 39-60 minutes, the lutein-administered group improved muscle tone significantly (P <0.05, Student's t-test) compared to the control group. 2 is a muscle tone measurement chart of rat soleus muscle to which astaxanthin or olive oil of Comparative Example 1 was orally administered. Expressed as mean ± standard error.

  Hereinafter, the present invention will be described in detail, but the present invention is not limited to the individual forms described below.

  The carotene oxidation derivative derived from land animals, land plants or microorganisms in the present invention is not particularly limited as long as it is derived from vegetables, fruits, microorganisms and the like containing carotenoids. Moreover, the carotene oxidation derivative of the present invention may contain at least one of a hydroxyl group, a carbonyl group, and an ether group. As an example of the carotene-oxidized derivative of the present invention, lutein is given and described below.

  Lutein is abundant in vegetables, fruits and plants. In particular, it is one of carotenoids widely present in the plant world, such as green-yellow vegetables such as kale, brussels sprouts, spinach, broccoli, and yellow flowers such as marigold. Among them, the petals of the Asteraceae are contained in high concentrations in petals and have been used since ancient times as feed for enhancing the coloration of egg yolks in poultry, and in recent years as food colorings that take advantage of their clear yellow color. It is used. Lutein is also known to improve eye diseases such as age-related macular degeneration and cataracts. The lutein CAS No. is 127-40-2, and lutein is also known as (3R, 3'R, 6'R) -β, ε-carotene-3,3'-diol ((3R, 3'R , 6'R) -β, ε-carotene-3,3'-diol), (3R, 3'R, 6'R) -3,3'-dihydroxy-β, ε-carotene ((3R, 3 ' R, 6′R) -3,3′-Dihydroxy-β, ε-carotene) or narrowly defined xanthophyll. Lutein belongs to the alcohol derivative of carotene. Lutein 20S (containing 20% by mass of lutein, Kyowa Hakko Bio) and the like are commercially available.

  Extraction of lutein is carried out by impregnating the above-mentioned plants and microorganisms with a solvent at room temperature or in a heated state, or by using a distillation apparatus such as steam distillation in addition to solvent extraction performed using an extraction device such as a Soxhlet extractor. Extraction method, supercritical extraction method using carbon dioxide gas in a supercritical state, or pressing method to obtain an extract by pressing, freeze-dried, and distilled water is added to remove the residue to obtain a supernatant A method or the like can be used. In addition, fruit juice or the like can be used after being subjected to treatment such as spray drying.

As the extraction solvent used for the above-described extraction, either a polar solvent or a nonpolar solvent can be used, and these can also be mixed and used. For example, water; alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as ethylene glycol, propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; tetrahydrofuran Linear and cyclic ethers such as diethyl ether; polyethers such as polyethylene glycol; halogenated hydrocarbons such as dichloromethane, chloroform and carbon tetrachloride; hydrocarbons such as hexane, cyclohexane and petroleum ether; benzene and toluene Aromatic hydrocarbons such as; pyridines; supercritical carbon dioxide; oils, waxes, other oils, and the like. These can be used alone or in combination of two or more, and can be repeated by changing the solvent. In .
Of these, water, ethanol, propylene glycol, butylene glycol and the like are preferably used, and a water / ethanol mixed solution is more preferably used.

  Examples of the means for separating and purifying the extract include activated carbon treatment, liquid-liquid distribution, column chromatography, liquid chromatography, gel filtration, and precision distillation.

In addition to lutein, examples of carotene oxidation derivatives of the present invention include carotene alcohol derivatives (zeaxanthin etc.), glycosides (crocin etc.), ether derivatives (spheroidene etc.), epoxide derivatives (violaxanthin etc.), aldehyde derivatives (torula) Rosinaldehyde, etc.) and carboxylic acid derivatives (such as torrurarosin), as well as apocarotenoids (crocetin, bixin, azafrine, etc.), secocarotenoids (β-carotenone, etc.), retro carotenoids (such as eschertuxanthin, rhodoxanthin), higher carotenoids (deca A carotene oxidation derivative belonging to, for example, prenoxanthine and the like, and those derived from land animals, land plants or microorganisms. For example,
Zeaxanthin (also called (3R, 3'R) -β, β-carotene-3,3'-diol, CAS No. 144-68-3, distributed in corn seeds, physalis, etc., Fluka, EXTRASYNTHESE SA Etc.)
Crocin (8,8'-Diapo-ψ, ψ-carotene-8,8'-dioic acid bis (6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester, CAS No, 42553 -65-1, distributed in saffron stigma, gardenia fruit, etc., available at Sigma / EXTRASYNTHESE SA etc.),
Spheroidene (also known as spheroidene, 3,4-Didehydro-1,2,7 ', 8'-tetrahydro-1-methoxy-ψ, ψ-carotene, CAS No. 13836-61-8, Rhodopseudomonas spheroides, etc.) distribution),
Violaxanthin (also called violaxanthin, 5α, 6α: 5'α, 6'α-Bisoxy-5,5 ', 6,6'-tetrahydro-β, β-carotene-3β, 3β'-diol, CAS No. 126 -29-4, petals of sansikisumire, distributed in many green leaves and fruits, available at CaroteNature, etc.),
Torralodin aldehyde (also called torularhodinaldehyde, distributed in Rhodotorula genus yeast),
Torralodin (also called torularhodin, 3 ', 4'-Didehydro-β, ψ-caroten-16'-oic acid, CAS No. 514-92-1, distributed in Rhodotorula genus yeast, etc., available at CaroteNature, etc.),
Crocetin (also referred to as crocetin, 8,8'-Diapo-ψ, ψ-carotene-8,8'-dioic acid, CAS No, 27876-94-4, saffron stigma, etc., available at EXTRASYNTHESE SA, etc.) ,
Bixin (also referred to as (9Z) -6,6'-Diapo-ψ, ψ-carotene-6,6'-dioic acid 6-methyl ester, CAS No. 6983-79-5, akinoki fruit) , Available at CaroteNature / ChromaDex, Inc., etc.)
Azafrin (5R, 6R) -5,6-Dihydro-5,6-dihydroxy-10'-apo-β, ψ-carotenoic acid, CAS No. 507-61-9・ Distributed to roots)
β-carotenone (also called β-carotenone, 5,6,5 ', 6'-Diseco-β, β-caroten-5,6,5', 6'-tetrone, distributed in Murraya exotica, etc., CaroteNature Etc.)
Eschscholtzxanthin (3S, 3'S) -4 ', 5'-Didehydro-4,5'-retro-β, β-carotene-3,3'-diol, CAS No. 472-73-1 , Distributed in petals etc.)
Rhodoxanthin (also known as rhodoxanthin, 4 ', 5'-Didehydro-4,5'-retro-β, β-carotene-3,3'-dione, CAS No. 116-30-3, yew fruit) , Available at CaroteNature, etc.),
Decaprenoxanthin (decaprenoxanthin, (2R, 2'R, 6R, 6'R) -2,2'-Bis [(E) -4-hydroxy-3-methyl-2-butenyl] -ε, ε-carotene CAS No. 28368-06-1, Flavobacterium dehydrogenans (bacteria), Arthrobacter glacialis (bacteria), etc.), but are not limited to these examples.

  The terrestrial animal, land plant or microorganism-derived carotene-oxidized derivative as the muscle tone improving agent, muscle fatigue reducing agent or muscle contracture preventing agent of the present invention is an extract or a separated fraction obtained by the method exemplified above. May be used as it is, as a diluted solution diluted with an appropriate solvent, or as a concentrated extract or dry powder, or may be prepared as a paste.

  The muscle tone improving agent of the present invention has an effect of reducing muscle tonus enhancement. Therefore, the muscle tone improving agent of the present invention can be used for reducing muscle fatigue and for preventing muscle contracture. As used herein, the term “muscle contracture” refers to “permanent and involuntary contraction of muscles or muscle groups with rigidity (supervised by Tomohiko Morioka, University of Medicine, Asakura Shoten, pp.300 ( 1985)) ”. In other words, it means that the state in which muscle tonus is abnormally increased continues and is caused by excessive load on the muscle, long-term fixation of the posture (due to work in a fixed posture, long-term medical treatment, joint inflammation, etc.), etc. It is. Further, in the present invention, “preventing muscle contracture” means to reduce the future increase in muscle tonus. Therefore, it is possible to prevent the occurrence of muscle contracture by reducing the abnormal increase in muscle tonus that will occur in the future. Note that muscle tonus is a constant muscle tension that skeletal muscles constantly hold, and has roles such as resistance to muscle extension, posture maintenance, and joint fixation (excessive movement inhibition). In the present invention, the degree of muscle tonus enhancement (muscle tonus enhancement degree) is referred to as “muscle tone degree”, and “improvement of muscle tone degree” refers to reducing muscle tonus enhancement. The contracted muscle causes further pain and inflammation due to deterioration of blood flow. The agent for preventing muscle contracture of the present invention can exert a preventive effect on these disorders. Conventionally, non-steroidal anti-inflammatory analgesics (ketoprofen) and the like have been used as drugs for improving muscle contracture.

The terrestrial animal, land plant or microorganism-derived carotene-oxidized derivative of the present invention significantly improves muscle tone due to electrical stimulation of the soleus muscle as compared to the control group (non-administration group), as shown in Examples below. That is, it significantly reduces muscle tonus enhancement, has the effect of reducing muscle fatigue and preventing muscle contracture. Therefore, land animals, land plants or microorganism-derived carotene oxidation derivatives can be used not only as muscle tone improving agents, but also as muscle fatigue reducing agents or muscle contracture preventing agents, Pharmaceuticals (or pharmaceutical composition) effective in improving muscle tone, reducing muscle fatigue, or preventing muscle contractures by combining these muscle tone improving agents, muscle fatigue reducing agents or muscle contracture preventing agents with other additives. It can also be used to produce food and drink. In addition, the pharmaceutical or the food or drink includes “a pharmaceutical composition or a food or drink used for improving muscle tone”, “a pharmaceutical composition or a food or drink used to reduce muscle fatigue”, “muscle restraint”. It can also be referred to as a “pharmaceutical composition or food or drink used for preventing shrinkage”. The agent for improving muscle tone, the agent for reducing muscle fatigue or the agent for preventing muscle contracture according to the present invention is a pharmaceutical product for humans or animals that exhibits an effect of improving muscle tone, an effect of reducing muscle fatigue or an effect of preventing muscle contracture, It can be used as quasi-drugs and foods. In addition, the present invention is based on the concept of improving endurance, anti-fatigue, and motor function in persons with exercise deficiency, middle-aged and elderly people, and bed resters, and foods, functional foods, foods for the sick, and special health use that indicate that effect. Can be applied to food. Furthermore, it can be used to cope with not only exercise deficient persons, middle-aged and elderly persons, bed rest persons, but also all people including athletes. The agent for improving muscle tone, the agent for reducing muscle fatigue or the agent for preventing muscle contracture according to the present invention includes a narrowing of the range of motion of joints, myofascial pain syndrome, myalgia, low back pain (muscle / fascia) Low back pain, degenerative spondylosis, intervertebral disc disease, lumbar sprain), stiff shoulders, fifty shoulders, osteoarthritis, shoulder periarthritis, tendon / tendon sheathitis, peritonitis, humerus condyle (such as tennis elbow), yes Muscle fatigue in oxygen exercise or muscle fatigue in anaerobic exercise can be reduced. The muscle tone improving agent, muscle fatigue reducing agent, or muscle contracture preventing agent of the present invention can prevent a decrease in physical function or bedridden in elderly people, fracture patients, long-term caregivers and the like.
The muscle tone improving agent of the present invention can also be expressed as follows.
1) A method for improving muscle tone, comprising a step of administering to a subject a pharmaceutically effective amount of a carotenylated derivative derived from a land animal, a land plant or a microorganism.
2) Use of carotene-oxidized derivatives derived from terrestrial animals, terrestrial plants or microorganisms in the production of muscle tone improving agents.
3) Land use animals, land plants, or microorganism-derived carotene-oxidized derivatives for use in improving muscle tone.
In the present invention, the improvement in the degree of muscle tone can also be expressed as a reduction in the increase of muscle tonus and the prevention of an increase in muscle tonus.
When the muscular tone improving agent of the present invention is used for therapeutic purposes, the muscular tone of the present invention is applied to a subject (for example, a subject with increased muscle tonus or a subject suspected to have increased muscle tonus). It is desirable to administer an improving agent.
On the other hand, when the muscular tone improving agent of the present invention is used for the purpose of prevention, the muscular tone improvement of the present invention is applied to a subject (for example, a subject suspected of having muscle tonus increased in the future) before the muscle tonus is increased. It is desirable to administer the agent.
The muscle fatigue reducing agent of the present invention can also be expressed as follows.
1) A method for reducing muscle fatigue, comprising the step of administering to a subject a pharmaceutically effective amount of a carotenylated derivative derived from a land animal, a land plant or a microorganism.
2) Use of carotene-oxidized derivatives derived from land animals, land plants or microorganisms in the manufacture of muscle fatigue reducing agents.
3) Land use animals, land plants, or microorganism-derived carotene-oxidized derivatives for use in reducing muscle fatigue.
In the present invention, reduction of muscle fatigue can also be expressed as prevention of muscle fatigue.
When the muscle fatigue reducing agent of the present invention is used for therapeutic purposes, the muscle fatigue reducing agent of the present invention is administered to a subject (for example, a subject in which the muscle is fatigued or a subject in which the muscle is suspected to be fatigued). It is desirable to do.
On the other hand, when the muscle fatigue reducing agent of the present invention is used for preventive purposes, the muscle tone improving agent of the present invention is administered to a subject (for example, a subject suspected of muscles becoming tired in the future) before the muscle fatigues. It is desirable to do.
The agent for preventing muscle contracture of the present invention can also be expressed as follows.
1) A method for preventing muscle contracture, comprising a step of administering to a subject a pharmaceutically effective amount of a carotenylated derivative derived from a land animal, a land plant or a microorganism.
2) Use of carotene-oxidized derivatives derived from terrestrial animals, terrestrial plants or microorganisms in the manufacture of preventive agents for muscle contracture.
3) Land use animal, land plant or microorganism-derived carotene derivative for use in preventing muscle contractures.
When the muscle contracture preventing agent of the present invention is used, the muscle contracture preventing agent of the present invention is administered to a subject (for example, a subject suspected of contracting muscle in the future) before the muscle contracts. Is desirable.
Examples of the administration target in the present invention include those with lack of exercise, middle-aged and elderly people, bed rest persons, athletes, long-term care persons, or narrowing of the range of motion of joints, myofascial pain syndrome, muscle pain, low back pain ( Muscle / fascial low back pain, deformed spondylosis, intervertebral disc disease, lumbar sprain), stiff shoulders, fifty shoulders, osteoarthritis, shoulder periarthritis, tendon / tendon sheathitis, peritonitis, suprascapular condylaritis (tennis elbow) Etc.), those who have muscle fatigue in aerobic exercise, muscle fatigue in anaerobic exercise, etc. can be exemplified, but are not limited thereto.

  Examples of the dosage form when the muscle tone improving agent, muscle fatigue reducing agent or muscle contracture preventing agent of the present invention is used as a pharmaceutical product or quasi-drug include tablets, capsules, granules, powders, syrups, etc. Oral administration or parenteral administration such as injections, suppositories, inhalants, transdermal absorption agents, external preparations and the like can be mentioned. Moreover, in order to prepare such various dosage forms, the lutein of the present invention alone or other pharmaceutically acceptable excipients, binders, extenders, disintegrants, surfactants , Lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents, and the like can be used in appropriate combinations. Of these dosage forms, the preferred form is oral administration, and when an oral liquid preparation is prepared, it can be produced by a conventional method by adding a flavoring agent, a buffering agent, a stabilizer and the like.

  The foods and drinks effective for improving muscle tone, reducing muscle fatigue, or preventing muscle contracture according to the present invention are not limited in category or type, and may be functional foods, foods for specified health use, health foods, and foods for nursing care. Alternatively, it may be a confectionery, a lactic acid bacteria beverage, a dairy product such as cheese or yogurt, or a seasoning. There is no restriction | limiting also about the shape of food / beverage products, It can take all the food / beverage product forms which can distribute | circulate normally, such as solid, liquid, liquid food form, jelly form, tablet form, granule form, and capsule form. The above-mentioned food and drink can be produced by ordinary methods of those skilled in the art. However, the terrestrial animal, land plant or microorganism-derived carotene oxide derivative of the present invention has an effect of improving muscle tone, reducing muscle fatigue or muscle contracture. Addition of carbohydrates, proteins, lipids, dietary fiber, vitamins, biologically essential trace metals (manganese sulfate, zinc sulfate, magnesium chloride, potassium carbonate, etc.), fragrances, and other ingredients as long as they do not interfere with the preventive action it can.

  In addition, the food and drink of the present invention specifically includes various foods and drinks, food and drink compositions (for example, milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crusts, prepared milk powder, and liquid foods. Foods for the sick, infant milk powders, lactating milk powders, nutritional foods, etc.), land animals and land plants having the effect of improving muscle tone, reducing muscle fatigue or preventing muscle contracture of the present invention Alternatively, a microorganism-derived carotene oxidation derivative may be added and ingested.

  The amount of the terrestrial animal, terrestrial plant or microorganism-derived carotene-oxidized derivative of the present invention relative to these varies depending on the form of use, but the terrestrial animal, terrestrial plant or microorganism-derived carotene-oxidized derivative of the present invention is lutein. An example of the case is as follows. In the form of food, it is usually 0.0001 to 10% by mass, more preferably 0.001 to 5% by mass, and particularly preferably 0.002 to 2% by mass. In the case of beverages, 0.001 to 0.5% by mass, 0.005 to 0.25% by mass, particularly 0.01 to 0.1% by mass is preferable. In the case of food tablets and / or capsules such as tablets, it is preferable that lutein is contained in an amount of 0.1 to 95% by mass, further 1 to 90% by mass, particularly 5 to 50% by mass. In addition, the state of the food or drink usually used, for example, solid (powder, granule, etc.), paste, liquid, suspension, or gel may be used.

  In the case of pharmaceuticals other than the above, such as oral solid preparations such as tablets, granules, capsules, and oral liquid preparations such as oral liquids and syrups, lutein is usually 0.01 to 95% by mass, Furthermore, it is preferable to set it as 5-90 mass%, especially 10-50 mass%.

  The other ingredients are not particularly limited, but the muscle tone improving agent, muscle fatigue reducing agent or muscle contracture preventing agent containing the carotenylated derivative derived from land animals, land plants or microorganisms of the present invention is used as a food or drink or food composition. When used in a product, water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like can be used as main components. Examples of the protein include whole milk powder, skim milk powder, partially skim milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin , Α-lactalbumin, lactoferrin, soy protein, chicken egg protein, meat protein and other animal and plant proteins, hydrolysates thereof; butter, whey minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipid, lactose, etc. And various milk-derived components. Examples of the saccharide include general saccharides, modified starch (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of the lipid include animal oils such as lard, fish oil, etc., fractionated oils, hydrogenated oil, transesterified oil, etc .; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, Examples include vegetable oils such as hydrogenated oils and transesterified oils. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. And minerals include, for example, calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals. Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid. These components can be used alone or in combination of two or more, and synthetic products and / or foods containing a large amount thereof may be used.

The dosage (effective intake) of the muscle tone improving agent, muscle fatigue reducing agent or muscle contracture preventing agent of the present invention is preferably 0.1 to 1500 mg / 60 kg body weight per day, and 1 to 1200 mg / 60 kg body weight. Is more preferable, and 5-1000 mg / 60 kg body weight is particularly preferable.
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

  Hereinafter, the present invention will be described with reference to experimental examples and examples, but the present invention is not limited thereto. In addition, in this specification,% display shows mass%, when not showing clearly.

[Experiment 1]
<Evaluation of ex vivo muscle tone improvement of lutein by rat soleus>
Method: The method of UCHIDA et al. (Masayuki UCHIDA et al, P2N-P9-1 Some affecting factors on the fatigue of the rat soleus muscle by electrical field stimulation, Journal of Pharmacological Sciences, 109 (Supplement I): 233, 2009 .).
Male SD rats (Japan SLC Co., Ltd.) were purchased at the age of 5 weeks, and animals (weight range: 170 to 190 g) in which no abnormalities were observed in observation of the general state were as similar as possible to the average weight of each group The body weight was stratified so as to be divided into 2 groups (each group n = 8) of lutein administration group and control group. 30 minutes after oral administration of the administered specimen, the rat was subjected to dissection, and the excised soleus muscle was placed in a Krebs solution at 30 ° C. (aeration conditions: 95% -oxygen, 5% -carbon dioxide) with a transducer (manufactured by Nihon Kohden Co., Ltd.). ) And a load of 0.5 g was applied. Thereafter, spasm caused by electrical stimulation (1.5 ms, 10 V, 2 Hz) was generated frequently and continuously, and the muscle was overloaded, and the contraction at that time was observed every minute. The muscle tension (g) when the muscle was relaxed at the time of each observation was measured as muscle tonus (g). Furthermore, FIG. 2 was created by calculating the muscle tone (g) at the time of each observation according to the following equation.
Muscle tone at each observation (g) = Muscle tonus at each observation (g)-Muscle tonus at the start of measurement (g)
The sample administered to the lutein administration group was prepared by suspending lutein 20S (containing 20% by mass of lutein, Kyowa Hakko Bio Co., Ltd.) in olive oil as a solvent to a concentration of 8 mg / mL. It was orally administered at a dose of 10 mL / kg. In the control group, the solvent was orally administered at a liquid volume of 10 mL / kg. The structural formula of lutein is shown below.

  The results are shown in FIG. As shown in FIG. 2, by repeating electrical stimulation, the muscle relaxation response gradually decreased and the muscle tone gradually increased in both groups. After 39 minutes of measurement, in the lutein administration group, the increase in muscle tone was suppressed significantly (P <0.05, Student's t-test) compared to the control group. In other words, it was found that lutein has an effect of reducing muscle fatigue and an effect of preventing the occurrence of muscle contracture.

[Comparative Example 1]
<Evaluation of improvement of ex vivo muscle tone by rat soleus>
Method: The method of UCHIDA et al. (Masayuki UCHIDA et al, P2N-P9-1 Some affecting factors on the fatigue of the rat soleus muscle by electrical field stimulation, Journal of Pharmacological Sciences, 109 (Supplement I): 233, 2009 .).
Male SD rats (Japan SLC Co., Ltd.) were purchased at the age of 5 weeks, and animals (weight range: 170 to 190 g) in which no abnormalities were observed in observation of the general state were as similar as possible to the average weight of each group The body weight was stratified so that it was divided into two groups (n = 8 for each group): an astaxanthin-administered group and a control group. 30 minutes after oral administration of the administered specimen, the rat was subjected to dissection, and the excised soleus muscle was placed in a Krebs solution at 30 ° C. (aeration conditions: 95% -oxygen, 5% -carbon dioxide) with a transducer (manufactured by Nihon Kohden Co., Ltd.). ) And a load of 0.5 g was applied. Thereafter, spasm caused by electrical stimulation (1.5 ms, 10 V, 2 Hz) was generated frequently and continuously, and the muscle was overloaded, and the contraction at that time was observed every minute. The muscle tension (g) when the muscle was relaxed at the time of each observation was measured as muscle tonus (g). Furthermore, FIG. 3 was created by calculating the muscle tone (g) at the time of each observation according to the following equation.
Muscle tone at each observation (g) = Muscle tonus at each observation (g)-Muscle tonus at the start of measurement (g)
The sample administered to the astaxanthin administration group was suspended in olive oil as a solvent in pure aster oil 80 (containing 8.0% by mass of astaxanthin, Yamaha Motor) to a concentration of 20 mg / mL, and astaxanthin was 16 mg / 10 mL. Orally administered at a dose of / kg. In the control group, the solvent was orally administered at a liquid volume of 10 mL / kg.

  The results are shown in FIG. As shown in FIG. 3, the astaxanthin-administered group did not improve muscle tone compared to the control group. That is, it was found that astaxanthin has no effect to prevent the occurrence of muscle contracture.

  The land animal, land plant or microorganism-derived carotene oxidant according to the present invention can improve muscle tone, reduce muscle fatigue or prevent muscle contracture. Further, the muscle tone improving agent, muscle fatigue reducing agent or muscle contracture preventing agent according to the present invention can be provided in various forms and can be easily used because it has less odor derived from raw materials. It can be provided as a food or drink or a medicine that improves muscle tone, reduces muscle fatigue, or prevents muscle contracture. Furthermore, since the present invention has no problem with respect to safety to the human body, the excellent effect can be easily and effectively utilized, and its utility value is high.

Claims (18)

A muscle tone improving agent comprising a carotene oxidation derivative derived from a land animal, a land plant or a microorganism as an active ingredient. The muscle tone improving agent according to claim 1, wherein the carotenylated derivative derived from land animals, land plants or microorganisms contains at least one of a hydroxyl group, a carbonyl group, and an ether group. The terrestrial animal, terrestrial plant or microorganism-derived carotene oxidation derivative is at least one or more of lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin The muscle tone improvement agent of Claim 1 or 2 which uses as an active ingredient. The muscle tone improving agent according to any one of claims 1 to 3, wherein an effective amount of the muscle tone improving agent is 0.1 to 1500 mg / 60 kg body weight. A pharmaceutical composition effective for improving muscle tone, comprising 0.01 to 95% by mass of the muscle tone improving agent according to any one of claims 1 to 4. A food and drink effective for improving muscle tone, comprising 0.0001 to 10% by mass of the muscle tone improving agent according to any one of claims 1 to 4. A muscle fatigue reducing agent comprising a carotene oxidation derivative derived from land animals, land plants or microorganisms as an active ingredient. The muscle fatigue-reducing agent according to claim 7, wherein the carotenylated derivative derived from land animals, land plants, or microorganisms contains at least one of a hydroxyl group, a carbonyl group, and an ether group. The terrestrial animal, terrestrial plant or microorganism-derived carotene oxidation derivative is at least one or more of lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin The muscle fatigue-reducing agent according to claim 7 or 8, comprising as an active ingredient. The muscular fatigue reducing agent according to any one of claims 7 to 9, wherein an effective amount of the muscular fatigue reducing agent is 0.1 to 1500 mg / 60 kg body weight. A pharmaceutical composition effective for reducing muscle fatigue, comprising 0.01 to 95% by mass of the muscle fatigue reducing agent according to any one of claims 7 to 10. A food and drink effective for reducing muscle fatigue, comprising 0.0001 to 10% by mass of the muscle fatigue reducing agent according to any one of claims 7 to 10. A muscular contracture preventive agent comprising a caroten-oxidized derivative derived from a land animal, a land plant or a microorganism as an active ingredient. The muscular contracture preventive agent according to claim 13, wherein the carotenylated derivative derived from land animals, land plants, or microorganisms contains at least one of a hydroxyl group, a carbonyl group, and an ether group. The terrestrial animal, terrestrial plant or microorganism-derived carotene oxidation derivative is at least one or more of lutein, zeaxanthin, crocin, spheroidene, violaxanthin, torularhodinaldehyde, torularhodin, crocetin, bixin, azafrin, β-carotenone, eschscholtzxanthin, rhodoxanthin, decaprenoxanthin The agent for preventing muscle contracture according to claim 13 or 14, comprising: as an active ingredient. The agent for preventing muscle contracture according to any one of claims 13 to 15, wherein an effective amount of the agent for preventing muscle contracture is 0.1 to 1500 mg / 60 kg body weight. A pharmaceutical composition effective for preventing muscle contracture, comprising 0.01 to 95% by mass of the agent for preventing muscle contracture according to any one of claims 13 to 16. A food and drink effective for preventing muscle contracture, comprising 0.0001 to 10% by mass of the agent for preventing muscle contracture according to any one of claims 13 to 16.

Images