WO2022045540A1 - Composition for improving muscle strength, alleviating muscle atrophy, or alleviating aging-induced sarcopenia by using ishige okamurae extract or compound isolated therefrom - Google Patents

Abstract

Disclosed in the present invention is a composition for improving muscle strength or alleviating muscle atrophy by using, in experiments on animal with muscle atrophy, modelsan Ishige okamurae extract or Ishophloroglucin A for alleviating hypotonia or muscle atrophy, promoting the expression of muscle protein synthesis-related factors Akt and PI3K or muscle growth-related factors TRPV4 and A1R, and inhibiting the expression of muscle protein synthesis inhibiting factors Myostatin and Sirt1 or muscle protein degradation-related factors MuRF1 and Atrogin1.

Description

Composition for improving muscle strength, improving muscle atrophy, or improving sarcopenia due to aging using a shellfish extract or a compound isolated therefrom

The present invention relates to a composition for improving muscle strength, improving muscle atrophy, or improving sarcopenia due to aging using an Ishige okamurae extract or a compound isolated therefrom.

The skeletal muscle is the largest tissue in our body, accounting for about 40-50% of the body weight of a normal person, and is responsible for essential functions such as movement, respiration, and heartbeat (Int J Biochem Cell Biol 37:1985-1996, 2005). ; Pharmacol Rev 61:373-393, 2009; Calcif Tissue Int 96:183-195, 2015). In the case of humans, the amount of muscle that is important for human activity starts to decrease by 1 to 2% each year after the age of 50, and by the age of 80, the amount of muscle decreases significantly to about half of the maximum muscle mass, and many elderly people suffer from sarcopenia (sarcopenia). ) or muscle atrophy, resulting in a decrease in physical function, disability, and deterioration of quality of life due to falls (Korean J Food and Nutr 4:133-139, 1991; J Nutr 127: 990S-991S, 1997; Curr Aging Sci 1:182-191, 2008) In particular, as the life expectancy increases and the proportion of the elderly increases rapidly, muscular atrophy is recognized as an important aging disease (Curr Opin Clin Nutr Metab Care). 13:1-7, 2010).

The muscle mass of the human body is normally maintained by balancing protein synthesis and degradation, but when protein degradation increases or synthesis decreases, muscle atrophy occurs. In addition to aging, nutritional deficiencies, and decreased activity, muscle atrophy is commonly observed in chronic wasting diseases such as cancer, diabetes, uremia, chronic lung disease, heart failure, and AIDS, but also occurs in acute diseases such as sepsis, surgery, trauma, and burns (Int J Biochem Cell Biol 37:2156-68, 2005).

The occurrence of muscle atrophy is related to the increase in the expression of proteins such as Atrogin-1 and Murf-1, which promote the breakdown of myofibrillar proteins, and the inhibition of the PI3K-Akt-mTOR signaling pathway involved in the synthesis of muscle-related proteins. It has been reported ( Am J Physiol Cell Physiol 287:C834-843, 2004). Specifically, the ubiquitin-proteasome system that decomposes muscle proteins including myosin is activated in muscle atrophy to restore muscle. It was confirmed that the decomposition of the constituent proteins was accelerated and, on the other hand, the activity of the PI3K-Akt-mTOR signaling pathway involved in muscle-related protein synthesis was lowered, resulting in inhibition of muscle protein synthesis ( Proc Natl Acad Sci USA 98). :14440-14445, 2001; J Biol Chem 280: 2737-2744, 2005). Therefore, in order to effectively inhibit muscle atrophy, not only suppress the signaling pathway related to the protein degradation pathway, but also up-regulate signaling pathways related to the biosynthesis of proteins essential for the increase of muscle mass and the activity of muscle cells. ) may be necessary.

The present invention is a shellfish extract, isophloroglucin A (Ishophloroglucin A) and diphlorethohydroxycarmalol (DPHC) isolated therefrom to improve muscle strength or muscle atrophy, sarcopenia due to aging start

It is an object of the present invention to provide a composition for improving muscle strength or muscle atrophy, and improving sarcopenia due to aging using a shellfish extract, isophloroglucin A and difluoroetohydroxycarmarol isolated therefrom.

Other objects or specific objects of the present invention will be set forth below.

As confirmed in the Examples and Experimental Examples below, the present inventors have isolated IPA (Ishophloroglucin A, Hexadecaphlorethol) of Formula 1 below (see Korean Patent No. 10-1964080, this patent document Considered a part of the present specification) and DPHC (Diphlorethohydroxycarmalol, Cas No 138529-[0001] 04-1) of Formula 2 below improve muscle weakness or muscle atrophy caused by dexamethasone in animal atrophy model animal experiments and are related to the synthesis of muscle proteins It was confirmed that it promotes the expression of Akt, PI3K, muscle growth-related factors, TRPV4, and A1R, and also inhibits the expression of Myostatin and Sirt1, muscle protein synthesis inhibitors, or MuRF1, Atrogin1, muscle protein degradation-related factors. Shell extract and DPHC improve muscle loss or sarcopenia caused by aging in animal models of aging-related sarcopenia, and promote the expression of muscle protein synthesis-related factors Akt and PI3K and muscle growth-related factors TRPV4 and A1R. It was confirmed that it inhibited the expression of Sirt1, a muscle protein synthesis inhibitor, or MuRF1, Atrogin1, a muscle protein degradation-related factor.

<Formula 1>

<Formula 2>

Considering the above, in one aspect, the present invention uses shellfish extract, isophloroglucin A, a compound of Formula 1, or DPHC, a compound of Formula 2, as an active ingredient. It can be identified as a composition for improving muscle strength or a composition for improving muscle atrophy, including, in another aspect, a shellfish extract, a compound of the <Formula 1>, isophloroglucin A (Ishophloroglucin A), or a compound of the <Formula 2> It can be identified as a composition for improving sarcopenia due to aging comprising phosphorus DPHC as an active ingredient.

As used herein, the term "shell extract" refers to a stem, leaf, fruit, flower, root, underground part, above-ground part, or a mixture thereof, which is an extraction target, water, a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), Methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or these An extract obtained by leaching using a mixed solvent, an extract obtained using a supercritical extraction solvent such as carbon dioxide or pentane, or a fraction obtained by fractionating the extract, and the extraction method considers the polarity of the active material, the degree of extraction, and the degree of preservation Therefore, any method such as chilling, reflux, heating, ultrasonic radiation, and supercritical extraction can be applied. In the case of the fractionated extract, the fraction obtained by suspending the extract in a specific solvent and mixing and standing still with a solvent having a different polarity, and adsorbing the crude extract to a column filled with silica gel, etc. It is meant to include the fraction obtained as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or solid extract from which the extraction solvent has been removed by methods such as freeze drying, vacuum drying, hot air drying, spray drying, and the like. Preferably, it refers to an extract obtained by using water, ethanol, or a mixed solvent thereof as an extraction solvent, and more preferably an extract obtained by using a mixed solvent of water and ethanol as an extraction solvent.

In addition, as used herein, the term "active ingredient" refers to a component that alone exhibits the desired activity or can exhibit activity together with a carrier that has no activity by itself.

Also in the present specification, "improving muscle strength" is meant to include muscle strength enhancement, muscle increase and/or muscle reduction inhibition.

In addition, as used herein, "improving muscular atrophy" is meant to include prevention, treatment, and alleviation of symptoms of muscular atrophy caused by aging, nutritional deficiency, reduced activity, cancer, diabetes, and the like.

Also, in the present specification, "improvement of sarcopenia due to aging" means recovery of muscle strength loss due to aging or recovery of muscle loss due to aging.

The composition of the present invention may contain the active ingredient in any amount (effective amount) as long as it can exhibit the intended muscle strength improvement activity, muscular atrophy improvement activity, aging-induced sarcopenia improvement activity, etc. depending on the specific use, formulation, product form, etc. However, a typical effective amount will be determined within the range of 0.001% to 15% by weight based on the total weight of the composition. As used herein, the term "effective amount" refers to when the composition of the present invention is administered to a mammal, preferably a human subject to the application, during the administration period as suggested by a medical professional, etc. It refers to the amount of the active ingredient included in the composition of the present invention that can exhibit the intended medical and pharmacological effects, such as effects. Such an effective amount can be determined empirically within the ordinary ability of one of ordinary skill in the art.

In a specific aspect, the composition of this invention can be grasped|ascertained as a food composition.

The food composition of the present invention can be prepared in any form, for example, beverages such as tea, juice, carbonated drinks, and ion drinks, processed oils such as milk and yogurt, gums, rice cakes, Korean sweets, bread, confectionery, noodles, etc. Foods, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and health functional food preparations such as bars can be manufactured.

In addition, the food composition of the present invention may have any product classification in terms of legal and functional classification as long as it conforms to the enforcement laws at the time of manufacture and distribution. For example, it is a health functional food according to Korea's "Health Functional Food Act", or confectionery, beans, tea, and beverages according to each food type in the Food Ordinance of Korea "Food Sanitation Act" (Ministry of Food and Drug Safety Notification "Food Standards and Specifications") , special purpose food, and the like.

The food composition of the present invention may contain food additives in addition to the active ingredients thereof. Food additives can be generally understood as substances that are added and mixed or infiltrated into food in manufacturing, processing, or preserving food. Food additives with guaranteed safety are limited in terms of ingredients or functions in the Food Additives Ordinance in accordance with the laws of each country that regulates the manufacture and distribution of food (“Food Sanitation Act” in Korea). In the Korean Food Additives Code (“Food Additive Standards and Specifications” announced by the Ministry of Food and Drug Safety), food additives are classified into chemically synthetic products, natural additives, and mixed preparations in terms of ingredients. It is divided into agents, preservatives, emulsifiers, acidulants, thickeners, etc.

The sweetener is used to impart appropriate sweetness to food, and both natural and synthetic ones may be used in the composition of the present invention. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents may be used to improve taste or aroma, and both natural and synthetic ones may be used. Preferably, it is a case where a natural thing is used. In the case of using a natural product, the purpose of nutritional enhancement in addition to flavor may be concurrently used. The natural flavoring agent may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, or the like, or obtained from green tea leaves, honeysuckle, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo can be used. The natural flavoring agent may be a liquid concentrate or a solid extract. Optionally, a synthetic flavoring agent may be used, and the synthetic flavoring agent may be an ester, an alcohol, an aldehyde, a terpene, or the like.

As a preservative, sodium calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, Pectin, etc. are mentioned, and acidulant, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. can be used as an acidulant. The acidulant may be added so that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing the taste.

As a thickening agent, a suspending agent, a settling agent, a gel former, a bulking agent, etc. can be used.

The food composition of the present invention may contain, in addition to the food additives described above, physiologically active substances or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutrition and guaranteed stability as food additives.

Examples of such physiologically active substances include catechins contained in green tea and the like, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, and the like. Minerals include calcium preparations such as calcium citrate, magnesium stearate Magnesium preparations, such as iron preparations, such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc, etc. are mentioned.

In the food composition of the present invention, the food additives as described above may be included in an appropriate amount to achieve the purpose of the addition according to the product type.

In relation to other food additives that may be included in the food composition of the present invention, reference may be made to the Food Ordinance of each country or the Food Additives Code.

The composition of the present invention may be regarded as a pharmaceutical composition in another specific embodiment.

The pharmaceutical composition of the present invention may be prepared as an oral dosage form or a parenteral dosage form according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the route of administration may be any suitable route including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of the combination of two or more routes is a case in which two or more formulations of drugs according to the route of administration are combined. For example, one drug is first administered by an intravenous route and the other drug is secondarily administered by a local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specifically, reference may be made to the pharmacopoeia of each country including the "Korea Pharmacopoeia".

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers according to methods known in the art together with a suitable carrier It can be prepared in a formulation such as Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Cellulose such as hydroxypropylmethylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, glycerol and the like. In the case of formulation, an appropriate binder, lubricant, disintegrant, colorant, diluent, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, and the like, and lubricants include sodium oleate. , sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium salt and calcium salt, polyethylene glycol, etc., and as a disintegrant, starch, methyl cellulose, agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt and the like. Examples of the diluent include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, and the like.

When the pharmaceutical composition of the present invention is prepared for parenteral use, it may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories together with suitable carriers according to methods known in the art. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or isotonic solution such as 5% dextrose may be used. . When formulated for transdermal administration, it can be formulated in the form of an ointment, a cream, a lotion, a gel, an external solution, a pasta agent, a liniment agent, an air roll, and the like. In the case of nasal inhalants, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, and the like. witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters, and the like can be used.

Specific formulations of pharmaceutical compositions are known in the art, and reference may be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). This document is considered a part of this specification.

A preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, severity of the patient, and the route of administration. It can be in the range /kg. Administration may be performed once or divided into several times a day. These dosages should not be construed as limiting the scope of the invention in any respect.

As described above, according to the present invention, a composition for improving muscle strength or muscle atrophy and improving sarcopenia due to aging using the shellfish extract, isophloroglucin A and difluoroetohydroxycarmarol isolated therefrom can provide

The composition of the present invention may be commercialized as food (especially health functional food) or medicine.

1 is a result showing the effect of shellfish extract, etc. on body weight and muscle weight in a muscle atrophy model.

2 is a result showing the effect of a plaque extract and the like on the leg weight in the muscle atrophy model.

3 is a result showing the effect of a plaque extract and the like on the grip strength in the muscle atrophy model.

4 is a result showing the effect of shell extract and the like on the weight and thickness of gastrocnemius in the muscle atrophy model.

5 is a result showing the effect of shellfish extract and the like on the mRNA expression of factors related to protein synthesis and degradation in gastrocnemius muscle in a muscle atrophy model.

6 is a result showing the effect of extracts of shellfish, etc. on the mRNA expression of factors related to the promotion and inhibition of muscle growth of gastrocnemius in a muscle atrophy model.

7 is a result showing the effect of shellfish extract and the like on the weight and thickness of soleus muscle in a muscle atrophy model.

8 is a result showing the effect of shellfish extract on the mRNA expression of factors related to protein synthesis and degradation of soleus muscle in a muscle atrophy model.

9 is a result showing the effect of shellfish extract and the like on the mRNA expression of factors related to promoting and inhibiting muscle growth of soleus muscle in a muscle atrophy model.

10 is a result showing the effect of shellfish extract and the like on the level of LDH (Lactate dehydrogenase) production in the muscle atrophy model.

11 is a result showing the effect of shellfish extract and DPHC on body weight, lean mass (muscle weight), etc. in a muscle atrophy model.

12 is a result showing the effect of plaque extract and DPHC on leg muscles in a muscle atrophy model.

13 is a result showing the effect of shellfish extract and DPHC on grip strength and endurance in a muscle atrophy model.

14 is a result showing the effect of shellfish extract and DPHC on the weight and thickness of gastrocnemius in a muscle atrophy model.

15 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of factors related to protein synthesis and degradation in gastrocnemius muscle in a muscle atrophy model.

16 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of factors related to promoting and inhibiting muscle growth in gastrocnemius in a muscle atrophy model.

17 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of muscle weakness inducing factor in gastrocnemius in a muscle atrophy model.

18 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of inflammation-related factors in gastrocnemius in a muscle atrophy model.

19 is a result showing the effect of shellfish extract and DPHC on the weight and thickness of soleus muscle in a muscle atrophy model.

20 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of factors related to protein synthesis and degradation of soleus muscle in a muscle atrophy model.

21 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of factors related to promoting and inhibiting muscle growth of soleus muscle in a muscle atrophy model.

22 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of muscle weakness inducing factor of soleus muscle in a muscle atrophy model.

23 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of inflammation-related factors in soleus muscle in a muscle atrophy model.

24 is a result showing the effect of shellfish extract and DPHC on body weight, lean mass (muscle weight), etc. in a sarcopenia aging model.

25 is a result showing the effect of a plaque extract and DPHC on leg muscles in a sarcopenia aging model.

26 is a result showing the effect of shellfish extract and DPHC on grip strength and endurance in the aging model of sarcopenia.

27 is a result showing the effect of shellfish extract and DPHC on the weight and thickness of gastrocnemius in an aging model of sarcopenia.

28 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of factors related to protein synthesis and degradation in gastrocnemius muscle in an aging model of sarcopenia.

29 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of factors related to the promotion and inhibition of muscle growth in gastrocnemius in an aging model of sarcopenia.

30 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of muscle weakness inducing factor in gastrocnemius in an aging model of sarcopenia.

31 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of inflammation-related factors in gastrocnemius in a sarcopenia aging model.

32 is a result showing the effect of shellfish extract and DPHC on the weight and thickness of soleus muscle in an aging model of sarcopenia.

33 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of factors related to protein synthesis and degradation of soleus muscle in a sarcopenia aging model.

34 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of factors related to promotion and inhibition of muscle growth in soleus muscle in a sarcopenia aging model.

35 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of muscle weakness inducing factor of soleus muscle in a sarcopenia aging model.

36 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of inflammation-related factors in soleus muscle in a sarcopenia aging model.

37 is a result showing the effect of shellfish extract and DPHC on the mRNA expression of LDH (Lactate dehydrogenase), a biomarker of sarcopenia in the aging model of sarcopenia.

Hereinafter, the present invention will be described with reference to Examples. However, the scope of the present invention is not limited to these examples.

<Example 1> Strength improvement or muscle atrophy improvement activity of shellfish extract and IPA (Ishophloroglucin A)

1. Material

Shellfish ( ishige okamurae , IO) collected in Seongsan-eup, Seogwipo-si, Jeju-do was decontaminated and dried, and 50% ethanol (alcohol) by weight of 10 times was added thereto, extracted at room temperature for 24 hours, filtered, and the extraction solvent was removed to prepare shellfish extract. did.

In addition, IPA was prepared by separating it according to the method described in the Examples of Korean Patent Registration No. 10-1964080.

2. Experimental method

2.1 Setting up the experimental group

After wearing 7-week-old ICR mice and acclimatizing them for 1 week, divide them into 7 groups as follows and measure their grip strength, and measure the weight of each group, muscles, leg muscles, gastrocnemius, soleus muscle, gastrocnemius and soleus muscle synthesis, growth, or decomposition or growth. An experiment was performed to measure the mRNA expression level of inhibitory factors.

① Group 1 (Saline administration group): Group administered saline once/day for 39 days

② Group 2 (Saline/Dexamethaone administration group): Administer saline once/day for 39 days, and subcutaneously dexamethaone (1mg/kg/day) for the last 10 days (29th to 38th). injection group.

③ Groups 3 to 5 (groups administered with shellfish extract 50, 100, and 200 mg/Kg/day): shellfish extract (50, 100, 200 mg/Kg/day) was administered once/day for 39 days, and the last 10 The group subcutaneously injected dexamethaone (1mg/kg/day) for daily (29th to 38th).

④ Group 6 (IPA) 3.5 mg/Kg/day group): IPA (3.5 mg/Kg/day) was administered once/day for 39 days, and dexamethaone (1 mg/kg/day) for the last 10 days (29th to 38th) ) subcutaneously. IPA is a Hexadecaphlorethol (HdP) compound isolated from plaque with Ishophloroglucin A (refer to Korean Patent No. 10-1964080)

⑤ Group 7 (oxymetholone 50 mg/Kg/day administration group): As a comparative drug, oxymetholone (50 mg/Kg/day), an oral muscle-increasing steroid, was administered once/day for 39 days, and the last The group subcutaneously injected with dexamethaone (1mg/kg/day) for 10 days (29th to 38th)

2.2 Determination of body weight and muscle weight

After conducting the test for 38 days, the animals used were weighed, sacrificed, and total muscle was collected and weighed.

2.3 Measurement of grip strength

For the measurement of the forelimbs using a grip strength meter, after confirming that the animal has grasped the bar with its toes, hold the tail base of the animal and keep the body horizontal and pull at a constant speed (2.5 cm per second) to release the bar The values indicated on the gauge were recorded. The measurement of the hindlimb is carried out in the same way as above by holding the animal's dorsal area and pulling it toward the tail. Each of the hind and forelimbs was measured and recorded three times, and the average value was evaluated.

2.4 Measurement of weight and thickness of gastrocnemius and soleus muscles

The gastrocnemius and soleus muscles among the leg muscles of the sacrificed animals were collected, weighed, and the diameter of the thickest thickness was measured several times, and the average value was used.

2.5 Measurement of mRNA expression levels of gastrocnemius and soleus muscle protein synthesis factors

After adding 1.0ml of Trizole to the muscle tissue crushed using a stick, and leaving it for 10 minutes at room temperature, 0.2ml of chloroform is added and vortexed vigorously for 15 seconds to completely dissolve (Lysis) made it This was left at room temperature for 10 minutes, and then centrifuged at 14,000 x g, 4°C for 15 minutes. The supernatant was transferred to a new tube, 0.5 ml isopropyl alcohol was added, and left at room temperature for 10 minutes, followed by centrifugation at 14,000 x g, 4°C for 10 minutes. After removing the supernatant, after washing the pellet with 1.0ml of 75% alcohol, 30 μl of DEPC-treated sterile distilled water was added to dissolve the pellet, and then used in subsequent tests.

Each isolated RNA was used as a template to synthesize cDNA according to the manufacturer's instructions using PrimeScript™ 1st strand cDNA Synthesis Kit. Using the cDNA as a template, a real-time polymerase chain reaction was performed as follows. Including oligonucleotide and SYBR Green I, real-time polymerase chain reaction was performed under the conditions of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and elongation at 72°C for 30 seconds.

3. Experimental results

3.1. Effect of plaque extract and IPA on muscle weight and leg muscle thickness in muscle atrophy model

Changes in body weight and muscle weight were confirmed compared to the group treated with shellfish extract and IPA, and the group treated with only dexamethasone, which induces muscle atrophy, for 10 days. As a result, as the significantly decreased body weight and muscle weight in the model inducing muscle atrophy increased significantly in the group administered with the sample and the group administered with oxymetholone as a comparative drug (p<0.01), shellfish extract It was confirmed that muscle atrophy caused by dexamethasone was inhibited by , IPA, and the comparative drug, oxymetholone (FIG. 1).

This change was also observed in the thickness change of the leg muscles. In the leg muscle thickness reduced by dexamethasone, a significant improvement effect was confirmed in the group administered with shellfish extract, IPA, and the comparative drug oxymetholone (FIG. 2).

3.2. Effect of plaque extract and IPA on grip strength in a muscle atrophy model

As a result of checking the grip strength to check whether it affects muscle strength as well as significant external muscle changes in the shell extract and IPA groups, it was confirmed that the grip strength decreased by muscle atrophy was significantly increased in the sample and the control group (Fig. 3). Accordingly, it was confirmed that the loss of grip strength could be prevented by significantly inhibiting the decrease in grip strength due to muscle atrophy by administering shellfish extract or IPA.

3.3. Effect of shellfish extract and IPA on gastrocnemius and soleus muscle in muscle atrophy model

In the muscle atrophy model, changes were confirmed in the gastrocnemius muscle, which is a fast muscle for agility, and the soleus muscle, a slow muscle for endurance. A significant decrease in gastrocnemius muscle was observed in the group induced by dexamethasone, and this decrease was significantly improved in the group administered with cncnfanf, IPA, and the comparative drug oxymetholone (Fig. 4). ). In particular, the weight and thickness of gastrocnemius muscle in the group administered with 100 and 200 mg/Kg/day shellfish extract improved to a level similar to that of the group administered with the comparative drug oxymetholone.

In addition, in the muscle atrophy model, the effect on the mRNA expression levels of Akt and PI3K, which are factors related to protein synthesis in gastrocnemius muscle, and the mRNA expression levels of MuRF1 and Atrogin1, which are factors related to degradation, was confirmed in the group administered with shellfish extract and IPA. As a result, it was confirmed that the mRNA expression of the gastrocnemius protein synthesizing factor increased and the mRNA expression of the degradation-related factor was decreased (FIG. 5). Accordingly, it was confirmed that the decreased gastrocnemius protein synthesis promotion and degradation inhibition in the muscle atrophy model could be improved by the administration of shellfish extract and IPA.

Also, in the muscle atrophy model, the effect on the mRNA expression level of TRPV4 and A1R, which are factors related to the promotion of muscle growth in gastrocnemius muscle, and the mRNA expression level of Myostatin and Sirt1, which are factors related to inhibition, was confirmed in the group treated with shellfish extract and IPA. As a result, it was confirmed that the mRNA expression of the gastrocnemius muscle growth promoting factor increased and the mRNA expression of the inhibitory factor decreased (FIG. 6). Accordingly, it was confirmed that the decreased gastrocnemius muscle growth promotion and inhibition inhibition in the muscle atrophy model could be improved by the administration of shellfish extract and IPA.

As such, it was confirmed that the gastrocnemius and soleus muscles were significantly improved by the administration of shellfish extract and IPA.

Also, significant changes in weight and thickness were observed in the soleus muscle, which is a slow muscle, by the administration of shellfish extract and IPA. It was confirmed that the decrease in soleus muscle due to dexamethasone was significantly inhibited in the shell extract administered groups at 100 and 200 mg/Kg/day (FIG. 7). In addition, it was observed that in the group administered with IPA, the weight and thickness of the soleus muscle could be protected at a level similar to that of the group administered with the comparative drug oxymetholone (FIG. 7). These results suggest that shellfish extract or IPA can inhibit the decrease in gastrocnemius caused by dexamethasone.

In addition, in the muscle atrophy model, the effect on the mRNA expression levels of Akt and PI3K, protein synthesis-related factors of soleus muscle, and the mRNA expression levels of MuRF1 and Atrogin1, which are degradation-related factors, was confirmed in the group administered with shellfish extract and IPA. It was confirmed that the mRNA expression of the factor related to the protein synthesis of the soleus muscle increased and the mRNA expression of the factor related to the degradation was decreased (FIG. 8). Accordingly, it was confirmed that the promotion of protein synthesis and degradation inhibition of soleus muscle protein, which was decreased in the muscle atrophy model, could be improved by the administration of shellfish extract and IPA.

In addition, in the muscle atrophy model, the effect on the mRNA expression levels of TRPV4 and A1R, which are factors that promote muscle growth in the soleus muscle, and the mRNA expression levels of Myostatin, Sirt1, which are inhibitory factors, was confirmed in the group administered with shellfish extract and IPA. It was confirmed that the mRNA expression of the soleus muscle growth promoting factor increased and the mRNA expression of the inhibitory factor decreased ( FIG. 9 ). Accordingly, it was confirmed that the promotion and inhibition of soleus muscle growth, which was decreased in the muscle atrophy model, could be improved by the administration of shellfish extract and IPA. Accordingly, the significant improvement ability of soleus muscle, the slow muscle that affects endurance, was confirmed by the shellfish extract and IPA.

3.4. Effect of shellfish extract and IPA on muscle strength improvement biomarkers in muscle atrophy model

Lactate dehydrogenase (LDH), an evaluation index of blood energy metabolism, decreased in the muscle atrophy model, but a significant increase was confirmed in the group administered with shellfish extract and IPA ( FIG. 10 ). LDH is used as an indicator of histological damage analysis and fitness evaluation of muscles according to exercise intensity, exercise duration, and recovery from fatigue, suggesting that muscle atrophy due to muscle atrophy can be suppressed by shellfish extract and IPA.

<Example 2> Strength improvement or muscle atrophy improvement activity of the preparation of shellfish extract and DPHC

1. Material

Shellfish ( ishige okamurae , IO) collected in Seongsan-eup, Seogwipo-si, Jeju-do was decontaminated and dried, and 50% ethanol (alcohol) by weight of 10 times was added thereto, extracted at room temperature for 24 hours, filtered, and the extraction solvent was removed to prepare shellfish extract. did

In addition, DPHC (Diphlorethohydroxycarmalol, Cas No 138529-04-1) was isolated and identified from the plaques ( ishige okamurae , IO) by the present inventors and stored by the present inventors.

2. Experimental method

2.1 Muscle atrophy model and its experimental group setting

After wearing 7-week-old ICR mice and acclimatizing them for 1 week, divide them into 7 groups as shown below to measure grip strength and endurance, and mRNA of related factors such as body weight, muscle, leg muscle, gastrocnemius, soleus muscle, and muscle synthesis or degradation of each group An experiment to measure the degree of expression was performed.

① Group 1 (Saline administration group): Group administered saline once/day for 39 days

② Group 2 (Saline/Dexamethaone administration group): Administer saline once/day for 39 days, and subcutaneously dexamethaone (1mg/kg/day) for the last 10 days (29th to 38th). injection group.

③ Groups 3 to 5 (groups administered with shellfish extract 50, 100, and 200 mg/Kg/day): shellfish extract (50, 100, 200 mg/Kg/day) was administered once/day for 39 days, and the last 10 The group subcutaneously injected dexamethaone (1mg/kg/day) for daily (29th to 38th).

④ Group 6 (DPHC) 2.5 mg/Kg/day administration group): DPHC (2.5 mg/Kg/day) was administered once/day for 39 days, and dexamethaone (1 mg/kg/day) for the last 10 days (29th to 38th) ) subcutaneously.

⑤ Group 7 (Decanoate 5 mg/Kg/day administration group): As a control drug, Decanoate (5 mg/Kg/day), an oral muscle-increasing steroid, was administered once/day for 39 days, and the last The group subcutaneously injected dexamethaone (1mg/kg/day) for 10 days (29th to 38th)

2.2 Determination of body weight and muscle weight

After conducting the test for 38 days, the animals used were weighed, sacrificed, and total muscle was collected and weighed.

2.3 Measurement of grip strength

For the measurement of the forelimbs using a grip strength meter, after confirming that the animal has grasped the bar with its toes, hold the tail base of the animal and keep the body horizontal and pull at a constant speed (2.5 cm per second) to release the bar The values indicated on the gauge were recorded. The hind limbs were measured in the same way as above by grabbing the animal's belly and pulling it toward the tail. Each of the hind and forelimbs was measured and recorded three times, and the average value was evaluated.

2.4 Ladder climbing

A weight corresponding to the weight of each animal was attached to the tail, and the time required to climb the ladder to the end was measured. Each was measured three times and recorded, and the average value was evaluated.

2.5 Measurement of weight and thickness of gastrocnemius and soleus muscles

The gastrocnemius and soleus muscles among the leg muscles of the sacrificed animals were collected, weighed, and the diameter of the thickest thickness was measured several times, and the average value was used.

2.6 Measurement of mRNA Expression Level

After adding 1.0ml of Trizole to the muscle tissue crushed using a stick, and leaving it for 10 minutes at room temperature, 0.2ml of chloroform is added and vortexed vigorously for 15 seconds to completely dissolve (Lysis) made it This was left at room temperature for 10 minutes, and then centrifuged at 14,000 x g, 4°C for 15 minutes. The supernatant was transferred to a new tube, 0.5ml isopropyl alcohol was added thereto, and left at room temperature for 10 minutes, followed by centrifugation at 14,000 x g, 4°C for 10 minutes. After removing the supernatant, after washing the pellet with 1.0ml of 75% alcohol, 30 μl of DEPC-treated sterile distilled water was added to dissolve the pellet, and then used in subsequent tests.

Each isolated RNA was used as a template to synthesize cDNA according to the manufacturer's instructions using PrimeScript™ 1st strand cDNA Synthesis Kit. Using the cDNA as a template, a real-time polymerase chain reaction was performed as follows. Including oligonucleotide and SYBR Green I, real-time polymerase chain reaction was performed under the conditions of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and elongation at 72°C for 30 seconds.

2.7 Statistical processing

The Kruskal-Wallis test and Mann-Whitney U post-hoc test were used to verify the significance between groups, and SPSS version 22 software (IBM Corporation, Armonk, NY, USA) was used to calculate significance ($$ p<0.01, $ p<0.05).

3. Experimental results

3.1 Effect of Plaque Extract and DPHC on Muscle Weight and Leg Muscle Thickness in Muscle Atrophy Model

Weight changes in body weight, fat mass, and lean mass were confirmed compared to the group treated with shellfish extract, DPHC, and the group treated with only dexamethasone, which induces muscle atrophy, for 10 days. As a result, no change was observed between the groups in body weight and fat mass, but as the lean mass in the muscle atrophy group (dexamethasone administered group) decreased significantly, the muscle loss in the muscle atrophy model was confirmed. Muscle loss was inhibited in the group administered with the shellfish extract and control drug (FIG. 11).

This change was also observed in the thickness change of the leg muscles. In the leg muscle thickness reduced by dexamethasone, there was a significant improvement effect in the group administered with the plaque extract, DPHC, and control drug (FIG. 12).

3.2 Effect of plaque extract and DPHC on grip strength and endurance in muscular atrophy model

As a result of checking grip strength and endurance to check whether muscle strength was affected as well as significant external muscle changes in the shellfish extract and DPHC group, grip strength and ladder climbing decreased by muscle atrophy were It was confirmed that the phosphorus shell extract, the DPHC administration group and the control drug administration group significantly increased (FIG. 13). Accordingly, it was confirmed that the loss of muscle strength could be prevented by significantly inhibiting the decrease in grip strength and endurance due to muscle atrophy by administering shellfish extract or DPHC.

3.3 Effect of plaque extract and DPHC on gastrocnemius in a muscle atrophy model

In the muscle atrophy model, recovery of muscle strength loss by administration of shell extract and DPHC samples was also observed in changes in the weight and thickness of gastrocnemius muscle. The gastrocnemius is a muscle that is used to produce a strong force, that is, a grip force. Significant improvement in the weight and thickness of gastrocnemius muscle reduced by dexamethasone was confirmed in all groups administered with the sample and control drug (FIG. 14).

In addition, in the muscle atrophy model, the effect on the mRNA expression levels of Akt and PI3K, which are protein synthesis-related factors of gastrocnemius muscle, and the mRNA expression levels of MuRF1 and Atrogin1, which are degradation-related factors, was confirmed in the group administered with shellfish extract and DPHC. As a result, it was confirmed that the mRNA expression of the gastrocnemius protein synthesizing factor increased and the mRNA expression of the degradation-related factor was decreased (FIG. 15). Accordingly, it was confirmed that the decreased gastrocnemius protein synthesis promotion and degradation inhibition in the muscle atrophy model could be improved by the administration of shellfish extract and DPHC.

In addition, in the muscle atrophy model, the effect on the mRNA expression level of TRPV4 and A1R, which are factors related to the promotion of muscle growth in gastrocnemius muscle, and the mRNA expression level of Sirt1, which is a factor related to inhibition, was confirmed. It was confirmed that the mRNA expression of the gastrocnemius muscle growth promotion-related factor increased and the mRNA expression of the inhibition-related factor decreased ( FIG. 16 ). Accordingly, it was confirmed that the decreased gastrocnemius muscle growth promotion and inhibition inhibition in the muscle atrophy model could be improved by the administration of shellfish extract and DPHC.

And, as a negative factor for muscle fiber growth and differentiation of gastrocnemius in the muscle atrophy model, the effect on the mRNA expression levels of Myostatin, TGF-β, and iNOS, which induce muscle weakness, was confirmed in the group administered with shellfish extract and DPHC. It was confirmed that the expression of the relevant mRNA was significantly reduced (FIG. 17). Accordingly, it was confirmed that unnecessary muscle loss could be suppressed by suppressing the muscle fiber weakening inducers excessively secreted from the gastrocnemius muscle of the muscle atrophy model by administration of the shellfish extract and DPHC.

In addition, changes in inflammation-related factors of gastrocnemius were observed in the muscle atrophy model. It was confirmed that the mRNA expression of IL-1β and TNF-α, which are inflammation-related factors of gastrocnemius, which were excessively increased in the muscle atrophy model, were significantly decreased in the group administered with the shellfish extract and DPHC ( FIG. 18 ).

As described above, it was confirmed that muscle strength could be improved by inducing significant improvement in gastrocnemius, a fast muscle that affects grip strength by shellfish extract and DPHC.

3.4 Effect of plaque extract and DPHC on soleus muscle in a muscle atrophy model

Significant changes in weight and thickness were also observed in the soleus muscle, which is a slow muscle, by the administration of shellfish extract and DPHC. The soleus muscle is used when the body needs balance and endurance. A significant improvement in the thickness of the soleus muscle reduced by dexamethasone was confirmed in all groups to which the sample and control drug were administered (FIG. 19). These results suggest that dexamethasone-induced degradation of soleus muscle can be inhibited by shellfish extract or DPHC.

In addition, in the muscle atrophy model, the effect on the mRNA expression of Akt and PI3K, which are factors related to protein synthesis in the soleus muscle, and the mRNA expression of MuRF1 and Atrogin1 related to degradation, was confirmed. It was confirmed that the mRNA expression of the factor increased and the mRNA expression of the degradation-related factor decreased ( FIG. 20 ). Accordingly, it was confirmed that the decreased protein synthesis promotion and degradation inhibition of soleus muscle in the muscle atrophy model could be improved by the administration of shellfish extract and DPHC.

As a result of confirming the effect on the mRNA expression of TRPV4 and A1R, which are factors related to promoting muscle growth in the soleus muscle in the muscle atrophy model, and the mRNA expression of Sirt1, an inhibitor related to the inhibitory factor, the group administered with the plaque extract and DPHC showed a significant association with the promotion of soleus muscle growth. It was confirmed that the mRNA expression of the factor was promoted and the mRNA expression of the inhibition-related factor was suppressed (FIG. 21). Accordingly, it was confirmed that the decreased soleus muscle growth and inhibitory ability in the muscle atrophy model could be improved by the administration of shellfish extract and DPHC.

And, as a negative factor for muscle fiber growth and differentiation of soleus muscle in the muscle atrophy model, the effect on the mRNA of Myostatin, TGF-β, and iNOS, which induce muscle weakness, was confirmed. It was confirmed that the expression of mRNA was significantly reduced (FIG. 22). Accordingly, it was confirmed that unnecessary muscle loss could be suppressed by suppressing the muscle fiber weakness inducing factors excessively secreted from the gastrocnemius muscle of the muscle atrophy model by administration of the shellfish extract and DPHC.

In addition, changes in inflammation-related factors of the soleus muscle were observed in the muscle atrophy model. It was confirmed that the mRNA expression of IL-1β and TNF-α, which are inflammation-related factors of soleus muscle, which were excessively increased in the muscle atrophy model, were significantly decreased in the group administered with the shellfish extract and DPHC ( FIG. 23 ). Accordingly, it was confirmed that muscle strength could be improved by inducing significant improvement in the soleus muscle, which is the slow muscle that affects grip strength, by using shellfish extract and DPHC.

<Example 3> Aging-induced sarcopenia improvement activity of shellfish extract and DPHC

1. Material

Shellfish ( ishige okamurae , IO) collected in Seongsan-eup, Seogwipo-si, Jeju-do was decontaminated and dried, and 50% ethanol (alcohol) by weight of 10 times was added thereto, extracted at room temperature for 24 hours, filtered, and the extraction solvent was removed to prepare shellfish extract. did

In addition, DPHC (Diphlorethohydroxycarmalol, Cas No 138529-04-1) was isolated and identified from the plaques ( ishige okamurae , IO) by the present inventors and stored by the present inventors.

2. Experimental method

2.1 Sarcopenia aging model and its experimental group setting

14-month-old B6 mice (mid to late 50s, female) were divided into 4 groups as follows to measure grip strength and endurance, and each group's weight, muscle, leg muscle, gastrocnemius muscle, soleus muscle weight, muscle synthesis or decomposition, etc. were measured. An experiment was performed to measure the mRNA expression level of the factor.

① Group 1 (Saline administration group): The group in which saline was administered once/day for 28 days.

② Group 2 (shell extract 100 mg/Kg/day administration group): Group administered shellfish extract (100 mg/Kg/day) once/day for 28 days.

③ Group 3 (DPHC) 2.5 mg/Kg/day administration group): The group administered DPHC (2.5 mg/Kg/day) once/day for 28 days.

④ Group 4 (Decanoate 5 mg/Kg/day administration group): As a control drug, an oral muscle increasing steroid, Decanoate (5 mg/Kg/day), was administered once/day for 28 days.

2.2 Determination of body weight and muscle weight

After performing the 28-day test, the utilized animals were weighed, sacrificed, and total muscle was collected and weighed.

2.3 Measurement of grip strength

For the measurement of the forelimbs using a grip strength meter, after confirming that the animal has grasped the bar with its toes, hold the tail base of the animal and keep the body horizontal and pull at a constant speed (2.5 cm per second) to release the bar The values indicated on the gauge were recorded. The hind limbs were measured in the same way as above by grabbing the animal's belly and pulling it toward the tail. Each of the hind and forelimbs was measured and recorded three times, and the average value was evaluated.

2.4 Ladder climbing

A weight corresponding to the weight of each animal was attached to the tail, and the time required to climb the ladder to the end was measured. Each was measured three times and recorded, and the average value was evaluated.

2.5 Measurement of weight and thickness of gastrocnemius and soleus muscles

The gastrocnemius and soleus muscles among the leg muscles of the sacrificed animals were collected, weighed, and the diameter of the thickest thickness was measured several times, and the average value was used.

2.6 Measurement of mRNA Expression Level

After adding 1.0ml of Trizole to the muscle tissue crushed using a stick, and leaving it for 10 minutes at room temperature, 0.2ml of chloroform is added and vortexed vigorously for 15 seconds to completely dissolve (Lysis) made it This was left at room temperature for 10 minutes, and then centrifuged at 14,000 x g, 4°C for 15 minutes. The supernatant was transferred to a new tube, 0.5ml isopropyl alcohol was added, and left at room temperature for 10 minutes, followed by centrifugation at 14,000 x g, 4°C for 10 minutes. After removing the supernatant, after washing the pellet with 1.0 ml of 75% alcohol, 30 μl of DEPC-treated sterile distilled water was added to dissolve the pellet, and then used in subsequent tests.

Each isolated RNA was used as a template to synthesize cDNA according to the manufacturer's instructions using PrimeScript™ 1st strand cDNA Synthesis Kit. Using the cDNA as a template, a real-time polymerase chain reaction was performed as follows. Including oligonucleotide and SYBR Green I, real-time polymerase chain reaction was performed under the conditions of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and elongation at 72°C for 30 seconds.

2.7 Statistical processing

The Kruskal-Wallis test and Mann-Whitney U post-hoc test were used to verify the significance between groups, and SPSS version 22 software (IBM Corporation, Armonk, NY, USA) was used to calculate significance ($$ p<0.01, $ p<0.05).

3. Experimental results

3.1 Effect of Plaque Extract and DPHC on Muscle Weight and Leg Muscle Thickness in Sarcopenia Aging Model

In the sarcopenia aging model Weight changes in body weight, fat mass and lean mass according to shell extract and DPHC administration were confirmed. As a result, the change in body weight between each group was not significant. However, in the group to which the sample was administered, the fat weight slightly decreased and the lean mass increased, but the results were not significant (FIG. 24).

But The thickness of the leg muscles showed an improvement effect in the group administered with the plaque extract, DPHC, and control drug (FIG. 25).

3.2 Effect of plaque extract and DPHC on grip strength and endurance in sarcopenia aging model

As a result of checking grip strength and endurance to check whether the administration of shellfish extract and DPHC affects muscle strength reduction as well as external muscle changes due to aging, the grip strength and ladder climbing decreased by aging were and it was confirmed that it was significantly increased in the control drug administration group (FIG. 26). Accordingly, it was confirmed that the decrease in grip strength and endurance due to aging could be significantly suppressed by administration of shellfish extract or DPHC, thereby preventing the loss of muscle strength.

3.3 Effect of plaque extract and DPHC on gastrocnemius in a sarcopenia aging model

In the aging model of sarcopenia, recovery of muscle strength loss by administration of shell extract and DPHC, which are samples, was also observed in changes in the weight and thickness of gastrocnemius muscle. The gastrocnemius is a muscle that is used to produce a strong force, that is, a grip force. A significant improvement in the weight and thickness of the gastrocnemius muscle reduced by aging was confirmed in all groups administered with the sample and control drug (FIG. 27).

In addition, in the sarcopenia aging model, the effect on the mRNA expression level of Akt and PI3K, protein synthesis-related factors of gastrocnemius muscle, and the mRNA expression level of MuRF1 and Atrogin1, which are degradation-related factors, was confirmed in the group administered with shellfish extract and DPHC. It was confirmed that the mRNA expression of the gastrocnemius protein synthesizing factor was increased and the mRNA expression of the degradation-related factor was decreased (FIG. 28). Accordingly, it was confirmed that the stimulation of gastrocnemius protein synthesis and inhibition of degradation caused by aging could be improved by administration of shellfish extract and DPHC.

In addition, as a result of confirming the effect on the mRNA expression level of TRPV4 and A1R, which are factors related to promoting muscle growth in gastrocnemius, and the mRNA expression level of Sirt1, an inhibitory factor, in the sarcopenia aging model, it was significant in the group administered with shellfish extract and DPHC. As a result, it was confirmed that the mRNA expression of the gastrocnemius muscle growth promoting factor increased and the mRNA expression of the inhibitory factor mRNA expression decreased (FIG. 29). Accordingly, it was confirmed that the stimulation and inhibition of gastrocnemius muscle growth decreased by aging could be improved by administration of shellfish extract and DPHC.

And, as a negative factor for muscle fiber growth and differentiation of gastrocnemius in an aging model of sarcopenia, the effect on the mRNA expression levels of Myostatin, TGF-β, GPX4, and iNOS, which induce muscle weakness, was confirmed. It was confirmed that the expression of related mRNA was significantly reduced in one group (FIG. 30). Accordingly, it was confirmed that unnecessary muscle loss could be suppressed by suppressing the muscle fiber weakening inducers excessively secreted from the gastrocnemius muscle due to aging by the administration of shellfish extract and DPHC.

In addition, changes in inflammation-related factors of gastrocnemius were observed in the sarcopenia aging model. It was confirmed that the mRNA expression of IL-1β and TNF-α, which are inflammation-related factors of gastrocnemius, which were excessively increased in the sarcopenia aging model, were significantly decreased in the group administered with the shellfish extract and DPHC ( FIG. 31 ).

As described above, it was confirmed that the improvement of sarcopenia caused by aging was possible as significant improvement of gastrocnemius was induced by shellfish extract and DPHC in the aging model of sarcopenia.

3.4 Effect of plaque extract and DPHC on soleus muscle in a sarcopenia aging model

In the sarcopenia aging model, significant changes in weight and thickness were also observed in the soleus muscle, which is a slow muscle, by administration of shellfish extract and DPHC. The soleus muscle is used when the body needs balance and endurance. Significant improvement in the thickness of the soleus muscle reduced by aging was confirmed in all groups to which the sample and control drug were administered (FIG. 32). These results suggest that the decline of soleus muscle due to aging can be inhibited by shellfish extract or DPHC.

In addition, as a result of confirming the effect on the mRNA expression of Akt and PI3K, which are factors related to protein synthesis of soleus muscle, and the mRNA expression of degradation-related MuRF1 and Atrogin1 in the aging model of sarcopenia, significant protein synthesis of soleus muscle in the group administered with the shellfish extract and DPHC It was confirmed that the mRNA expression of the related factor increased and the mRNA expression of the degradation related factor decreased ( FIG. 33 ). Accordingly, it was confirmed that the promotion of protein synthesis and inhibition of degradation of the soleus muscle reduced by aging could be improved by administration of shellfish extract and DPHC.

In addition, as a result of confirming the effect on the mRNA expression of TRPV4 and A1R, which are factors related to promoting muscle growth in the soleus muscle, and the mRNA expression of Sirt1, a factor related to inhibition, in the sarcopenia aging model, there was a significant difference in the group administered with the plaque extract and DPHC. It was confirmed that mRNA expression of growth-promoting factors was promoted, and mRNA expression of inhibitory factors was suppressed (FIG. 34). Accordingly, it was confirmed that the growth and inhibition of soleus muscle muscle growth and inhibitory ability decreased by aging could be improved by administration of shellfish extract and DPHC.

And, as a negative factor for muscle fiber growth and differentiation of soleus muscle in the aging model of sarcopenia, the effect on the mRNA of Myostatin, TGF-β, GPX4, and iNOS, which induce muscle weakness, was confirmed. As a result, the group administered with shellfish extract and DPHC. It was confirmed that the mRNA expression of the related factor was significantly reduced (FIG. 35). Accordingly, it was confirmed that unnecessary muscle loss could be suppressed by inhibiting the muscle fiber weakness inducers excessively secreted from the gastrocnemius muscle due to aging by the administration of shellfish extract and DPHC.

In addition, changes in inflammation-related factors of soleus muscle were observed in the sarcopenia aging model. It was confirmed that the mRNA expression of IL-1β and TNF-α, which are inflammation-related factors of soleus muscle, which were excessively increased by aging, were significantly decreased in the group administered with the shellfish extract and DPHC ( FIG. 36 ).

As described above, it was confirmed that the improvement of sarcopenia caused by aging was possible by inducing significant improvement in the soleus muscle, which is the slow muscle that affects the grip strength, by the shellfish extract and DPHC.

3.5. Effect of plaque extract and DPHC on sarcopenia biomarkers in sarcopenia aging model

As a result of confirming the mRNA expression level of LHD (Lactate dehydrogenase), a biomarker for muscle strength and muscle wasting in the blood, it was observed that LHD, an evaluation index of muscle strength, was significantly increased by shellfish extract and DPHC in the sarcopenia aging model (FIG. 37) ).

Claims (12)

1. A composition for improving muscle strength comprising shellfish extract, isophloroglucin A (Ishophloroglucin A) or diphlorethohydroxycarmalol as an active ingredient.
2. According to claim 1,

   The extract is a composition, characterized in that the extract obtained by extraction with water, ethanol, or a mixed solvent thereof.
3. 3. The method of claim 1 or 2,

   The composition is a pharmaceutical composition, characterized in that the composition.
4. 3. The method of claim 1 or 2,

   The composition is a food composition, characterized in that the composition.
5. A composition for improving muscular atrophy comprising a shellfish extract, isophloroglucin A (Ishophloroglucin A) or diphlorethohydroxycarmalol as an active ingredient.
6. 6. The method of claim 5,

   The extract is a composition, characterized in that the extract obtained by extraction with water, ethanol, or a mixed solvent thereof.
7. 7. The method according to claim 5 or 6,

   The composition is a pharmaceutical composition, characterized in that the composition.
8. 7. The method according to claim 5 or 6,

   The composition is a food composition, characterized in that the composition.
9. A composition for improving sarcopenia due to aging comprising shellfish extract or diphlorethohydroxycarmalol as an active ingredient.
10. 10. The method of claim 9,

    The extract is a composition, characterized in that the extract obtained by extraction with water, ethanol, or a mixed solvent thereof.
11. 11. The method of claim 9 or 10,

    The composition is a pharmaceutical composition, characterized in that the composition.
12. 11. The method of claim 9 or 10,

    The composition is a food composition, characterized in that the composition.

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