KR20200008332A - Composition for strengthening of muscle, preventing and treating Sarcopenia comprising Agarum cribrosum - Google Patents

Abstract

The present invention relates to a composition for muscle strengthening or preventing and treating sarcopenia, containing an Agarum cribrosum extract. The Agarum cribrosum extract of the present invention has no cytotoxicity and has excellent anti-myostatin activity, thereby being able to be usefully used as a pharmaceutical composition for muscle strengthening, bone growth induction, or preventing and treating sarcopenia.

Description

Composition for strengthening of muscle, preventing and treating Sarcopenia comprising Agarum cribrosum

The present invention relates to a composition for the prevention and treatment of muscle strengthening or myopathy, including pore seaweed extract.

In the 20th century, the number of populations has doubled, but in the 21st century, it is difficult to double the growth of the population due to lower birth rates and longer life expectancy. According to the 2015 UN's World population prospects (the 2015 revision), the population over 60, from 910 million in 2015, will grow to about 1.4 billion by 2030, to 2.1 billion by 2050. It is estimated. In addition, the elderly aged 80 or older will reach about 438 million by 2050, and this increase is expected to continue for decades to come. In Korea, the population aged 65 and over reached 7.2% of the total population in 2000 and entered the aging society.In 2018, the aged population reached 20.8%. The National Statistical Office expects to enter a post-aged society. In addition, it is pointed out as a big problem that Korea is approaching a very fast time to reach an aging society or an aging society compared to other countries. This problem is thought to be caused by the recent rapidly lower fertility rate, resulting in a sharp increase in the number of elderly population.

Aging is a phenomenon in which the physical function of living organisms deteriorates over time. In humans, when they are 80 years old, hearing is reduced by 30%, blood output by 45%, lung capacity by 60%, and muscle mass by 50%. have. Various physiological activities are not reduced by aging and some enzyme activity or hormone secretion function is increased. Therefore, the physiological change due to aging is a defect of the target functional enhancement or control mechanism, so it can be said that the adaptation ability is not reduced, but the physiological change.

Representative physiological changes with age are a decrease in muscle mass and strength. It is known that the decrease in muscle mass occurs after about 40 years, and about 8% decrease occurs in 10 years until the 70s, and thereafter, a sharp decrease occurs and occurs up to 15% in 10 years. Sarcopenia in the elderly causes a direct decrease in muscle strength, which increases the risk of death due to decreased physical function and impairment, as well as reduced metabolism and reduced immunity, such as high blood pressure, diabetes, arthritis, obesity, cancer, etc. It is also a cause of increasing the prevalence of metabolic diseases.

In connection with such a decrease in muscle mass, studies of myostatin that inhibits the growth of skeletal muscle have been made, and antibodies for inhibiting the function of myostatin have been prepared. However, side effects have appeared, and antimyostatin materials using natural products Although research is ongoing, most studies have been conducted at the cellular level, and few studies have reached clinical trials.

Korean Patent Publication No. 10-2005-0091015 Korean Patent Publication No. 10-2013-0069188

It is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of muscle strengthening or myopathy, including a hole seaweed extract.

Another object of the present invention to provide a health functional food for the prevention and improvement of muscle strengthening or myotropia containing a hole seaweed extract.

In addition, another object of the present invention is the step of extracting the alcohol added to the dried seaweed seaweed; Concentrating the extract under reduced pressure to remove the solvent; It is to provide a method for producing a composition for preventing and treating muscle strengthening and myotropia comprising the step of recovering the supernatant obtained by centrifuging the obtained pore seaweed extract.

In addition, another object of the present invention is to provide a feed additive comprising a hole seaweed extract.

The inventors of the present invention, while conducting various studies to develop a natural material for preventing and treating muscular dystrophy by strengthening muscle strength or aging, confirmed that the persimmon seaweed extract is active against antimyostatin. Muscle strengthening, bone growth induction or myopathy treatment of the extract is not known at all until now, it can be said that the significance is very significant in the first development by the present inventors.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention and treatment of muscle strengthening, bone growth induction or myopathy, including a puncture seaweed extract.

In one embodiment of the present invention, the composition has antimyostatin activity, improves muscle strength and muscle density, and has an effect of increasing muscle mass, bone weight, bone volume, and bone density.

In one embodiment of the present invention, the muscular dystrophy may be muscular dystrophy, myasthenia gravis, muscular dystrophy, myopathy, myotonia, muscle weakness, muscle regression axis, muscular dystrophy or myasthenia gravis.

In one embodiment of the present invention, the perforated seaweed extract is an organic solvent extract, preferably water or alcohol extract, more preferably an ethanol extract.

As another aspect, the present invention provides a functional food for preventing and alleviating myopathy, including the geunjeol seaweed extract as an active ingredient.

In still another aspect, the present invention provides a health functional food for strengthening muscle strength, which includes an extract of poultry wakame as an active ingredient.

In another aspect, the present invention comprises the steps of extracting by applying hot water to the dried seaweed seaweed; Concentrating the extract under reduced pressure to remove the solvent; It provides a method for producing a composition for preventing and treating myotropenia or strengthening muscle strength, comprising the step of recovering the supernatant by centrifuging the obtained pore seaweed extract.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating myotropia or a food for preventing and alleviating myopathy, comprising the extract of perilla sesame seaweed prepared by the preparation method.

In still another aspect, the present invention provides a feed additive comprising a hole seaweed extract as an active ingredient.

Since the persimmon seaweed extract of the present invention is not cytotoxic and has an excellent effect on the activity of antimyostatin, as a result, it can be used for the prevention, alleviation and treatment of senile muscle diseases such as myopathy.

Figure 1 shows a process for preparing a hole seaweed extract.
Figure 2 shows the results of the cytotoxicity test according to the concentration of the oleracea extract.
Figure 3 shows the results of the antimyostatin activity, Activin A and GDF11 inhibitory activity of the oleracea extract.
Figure 4 shows the results of the myostatin signaling inhibition experiment of pore seaweed extract.
Figure 5 shows the results of measuring weight and muscle strength after the treatment of poultry wakame extract in animal models.
Figure 6 shows the cardiopulmonary endurance and muscular endurance measurement results after the treatment of poultry wakame extract in animal models.
Figure 7 shows the results of the measurement of muscle mass, muscle volume and muscle density after the treatment of poultry wakame extract in animal models.
Figure 8 shows the bone weight, bone volume and bone density measurement results after the treatment of poultry wakame extract in animal models.
Figure 9 shows the results of blood analysis after the treatment of poultry wakame extract in animal models.
Figure 10 shows the organic solvent fractionation process of ethanol extract of poultry seaweed.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for preventing or treating myopathy, including Agarum cribrosum extract for preventing and treating myotropenia due to muscle strengthening or aging.

Agarum cribrosum is a yearly temperate species belonging to the Kelpaceae family. It is an algae and is also known as Yagishiri seaweed seaweed. It is mainly distributed in Japan's Hokkaido, Kuril Islands, the Americas Pacific Coast, and Bering Sea. Except for the thick leaf vein in the middle of the leaf, all the leaves have many holes, the roots are thin fibrous, and the stem is columnar to oblate. Inhabits all year long in the depths of 30 m. It contains many carbohydrates and is used as a raw material for alginic acid and potassium. It also contains a large amount of dietary fiber and is rich in natural minerals such as K, Ca, Na, and Mg and essential amino acids. The extract of perilla seaweed is very effective in antioxidation due to a large amount of physiologically active substances, has the effect of preventing skin aging and improving skin wrinkles, and is used as a cosmetic ingredient by acting as a natural preservative. Alginate, a polysaccharide component, increases food stagnation time in the stomach to prevent fasting, blocks fat absorption, and binds cholesterol or sodium in the intestine to be released into the body to lower cholesterol and blood pressure, thereby preventing and improving obesity, hyperlipidemia and dyslipidemia. It is effective in preventing constipation symptoms. Recently, it has been known to have an effect of increasing the concentration of acetylcholine, which is effective in Alzheimer's disease caused by neurodegeneration.

As used herein, the term "strength strengthening" means an increase in muscle strength, muscle density and bone density.

As used herein, the term "myotropenia" refers to a disease in which the muscle volume and strength gradually decline.

In the present invention, the kind of the extraction solvent used to extract the hole seaweed is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent may include water, alcohols having 1 to 4 carbon atoms or mixed solvents thereof, and these may be used alone or in combination of one or more thereof. Preferably water or ethanol may be used, more preferably ethanol, but is not limited thereto. The ethanol may be 1 to 100%, but is not limited thereto. In the present invention, the extract may be a hydrothermal extract or ethanol extract.

The extract has anti myostatin activity.

That is, the present invention relates to a composition for preventing and treating myopathy comprising an extract of pomegranate as an active ingredient, and the composition includes a pharmaceutical composition and a food composition. Sarcopenia is a disease caused by a decrease in skeletal muscle mass, which is known to directly lead to a decrease in muscle strength, resulting in various physical functions and disorders, and also increases the risk of death. It is a disease that results from gradual skeletal muscle loss associated with aging (Park, Sung-Won, 2007, Muscle Reduction of the Elderly, Korean Journal of Endocrinology, Vol. 22, No. 1). In particular, the puncture seaweed extract of the present invention specifically inhibits myostatin activity, which is a factor that inhibits the increase in muscle mass, and has a therapeutic and prophylactic effect for muscle reduction.

In addition, the present invention is directed to a composition for strengthening muscles comprising the extract of pomegranate seaweed as an active ingredient, the composition comprises a health food composition or a food composition. "Increasing muscle strength" refers to a phenomenon in which overall muscle strength increases due to an increase in skeletal muscle mass, an improvement in muscle function, or an increase in bone density, and does not mean a limited effect on a patient group of a specific disease.

As used herein, "Myostatin (MSTN)" is a protein that regulates muscle growth, belongs to the transforming growth factor-β TGF-β family and grows and differentiation factor-8 (GDF-8). )to be.

It is known that myostatin binds directly to activin receptor type 2 and also affects Smad signaling mechanisms. Inhibiting the activity of myostatin is known to promote muscle differentiation and play an important role in the improvement of various metabolic diseases. In the present invention, by adjusting the myostatin activity to improve the symptoms of muscle diseases such as myotropia, and can strengthen muscle strength, pharmaceutics including pomegranate extract that can improve bone weight, bone volume and bone density Provided are compositions and food compositions.

The composition of the present invention may be prepared as a pharmaceutical composition. The pharmaceutical composition of the present invention may be administered orally or parenterally, and preferably applied by oral administration. Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.001-100 mg / kg on an adult basis.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, water It is prepared by mixing cross or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solvents, emulsions, and syrups. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

On the other hand, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects. The severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and rate of release, duration of treatment, factors including the drug used in combination or simultaneous use, and other factors well known in the medical arts may be determined. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

The dosage of the pharmaceutical composition of the present invention can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the age, weight, sex, history, or type of substance used as an active ingredient of the patient. For example, the pharmaceutical composition of the present invention may be administered at about 0.1 ng to about 100 mg / kg, specifically, 1 ng to about 10 mg / kg per adult, and the frequency of administration of the composition of the present invention is particularly limited thereto. However, it may be administered once a day or several times in divided doses. However, since the dosage may vary depending on the route of administration, the severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

In another aspect, the present invention provides a health functional food for the prevention and improvement of muscle strength or myotropia comprising a hole seaweed extract.

The food composition of the present invention includes compositions for all types of foods, such as functional foods, nutritional supplements, health foods and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art. For example, the health food can be prepared in the form of gum, vitamin complex, tea, juice and drink containing the persimmon seaweed extract, granulating, encapsulating or powdering a food composition containing the persimmon seaweed extract as an active ingredient. It can be consumed by becoming angry. The functional food composition of the present invention may include ingredients that are commonly added in food production, and may include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared with a drink, in addition to the pore seaweed extract of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract, licorice extract may be further included.

The food composition includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and salts thereof, organic acids , Protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. It may also contain extracts for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination.

In another aspect, the present invention provides a feed composition comprising an extract of poultry seaweed as an active ingredient. The persimmon seaweed extract may be prepared in the same manner as in the pharmaceutical composition or food composition.

The horsetail seaweed extract of the present invention may be included in the feed composition as a growth promoter of an animal or a livestock because it exhibits an overall increase in strength due to an increase in skeletal muscle mass or an improvement in muscle function and bone density.

Specifically, the feed comprising a hole sesame seaweed extract according to the present invention as an active ingredient can be prepared in a variety of forms known in the art, preferably rich feed, irradiated feed or special feed may be included.

The rich feed includes seed fruits and grains containing grains such as wheat, oats and corn, and bran, soybeans, fluids, sesame seeds, linseed and coco palms, including rice bran, wheat bran and barley bran. Concentrated fresh liquids obtained from fish oil, fish tails, and fish residues such as by-products such as oil products, sweet potatoes, potatoes, etc. Animal feed and yeast such as dried whey, which is the balance of fish soluble, fish soluble, meat meal, blood meal, milk powder, skim milk powder, milk from cheese, and casein from skim milk , Chlorella and algae.

Forage, raw vegetables such as weeds, grasses, and green onions, fodder turnips, fodder beets, and root vegetables such as Lutherbearger, a type of turnip, raw grass, green onion crops, and grains Silage (silage), grasses and grasses that are dried and fertilized, lactic acid fermented storage feed, straw of breeder crops, and leaves of legumes.

Special feeds contain mineral feeds such as shells and rock salts, urea feeds such as urea and its derivatives such as diureide isobutane, and natural feed ingredients, which are not likely to be insufficient. Feed additives, dietary supplements, and the like are added to the.

The feed composition according to the present invention may comprise various feed additives.

In the present invention, the term "feed additive" refers to a substance added to a feed for various effects such as supplementation of nutrients and prevention of weight loss, enhancement of digestive availability of fiber in the feed, improvement of oil quality, prevention of reproduction disorder, improvement of conception rate, prevention of high temperature stress in summer, and the like. Say. The feed additive of the present invention corresponds to a feed supplement under the Feed Control Act, and minerals such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals, and trace minerals such as zinc, copper, cobalt, and selenium. Preparations, vitamins such as keratin, vitamin E, vitamins A, D, E, nicotinic acid, and vitamin B complexes, protective amino acids such as methionine and lyric acid, protective fatty acids such as fatty acid calcium salts, probiotics (lactic acid bacteria), yeast culture Probiotics such as water, mold fermentation products, yeasts and the like may be further included.

The feed composition according to the present invention is not particularly limited as long as it is an individual for the purpose of increasing the total muscle strength, increasing the bone mineral density and promoting growth due to an increase in the amount of skeletal muscle or an improvement in muscle function, and an increase in bone density. Such individuals include animals such as non-primates (eg, cattle, pigs, horses, cats, dogs, rats and mice) and primates (eg, monkeys, such as cynomolgous monkeys) and Mammals, including chimpanzees). In another embodiment, the subject is a livestock animal (eg horse, cow, pig, etc.) or a pet animal (eg dog or cat). In addition, fish, for example, flounder, flounder, eel, freshwater eel, eel, rockfish.

The dosage of the feed composition according to the invention will depend on a number of factors such as the species, size, weight and age of the individual. In principle, typical dosages can range from 0.001 to 10 g per individual / day. However, the present invention is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

<Example 1> Preparation of puncture seaweed extract

After thoroughly drying the sea bream seaweed was extracted with ethanol at a concentration of 20 mg / ml. Ethanol extraction was added 1ml of 100% ethanol to 20mg of dried seaweed seaweed and extracted three times at room temperature, dark for 24 hours. The extracted sample was removed from the ethanol using a rotary vacuum concentrator, centrifuged at 4000 rpm for 20 minutes, and filtered with a 0.2 um pore size filter to recover only the supernatant. The collected supernatant was measured for concentration of the sample using a centrifugal evaporator. As a result of three repeated extractions, an extraction yield of 1.20% was confirmed (FIG. 1).

Example 2 Cytotoxicity of Algae Extract

The cytotoxicity of Agarum cribrosum ethanol extracts (ACEE) prepared in Example 1 was confirmed. For cytotoxicity experiments, HEK293 cells per well in 96 well plates were seeded in 1 × 10 ⁴ cell / 100 ul (in DMEM with 10% FBS and 1% penicillin / streptomycin) cells for 24 hours in a 5% CO ₂ incubator. Thereafter, the hole seaweed ethanol extract (ACEE) was treated by concentration (100, 10, 1, 0.1 ug / ml) and incubated for 12 hours in a 5% CO ₂ incubator. Thereafter, 10 ul of WST reagent (DaeilLab, Korea) was added and the reaction was carried out for 1 hour, and then measured for absorbance at 415 nm to determine cytotoxicity. Positive control group was not treated with any cell, negative control group was treated with H ₂ O ₂ to induce apoptosis. Cytotoxicity was calculated by the following formula.

As a result, oleracea ethanol extract (ACEE) did not show cytotoxicity at a concentration of 100 ug / ml or less in HEK293 cells (Fig. 2).

Example 3 Antimyostatin Activity of Pork Seaweed Extract

The antimyostatin activity of the horsetail seaweed ethanol extract (ACEE) prepared in Example 1 was confirmed through a luciferase assay system.

Antimyostatin activity experiments were performed by seeding HEK293 cells in DMEM medium (10% FBS, 1% penicillin / streptomycine, 1% geneticine) in 96 well plates at 2.0 × 10 ⁴ cells per well for 24 hours in a 5% CO ₂ incubator. It was. Subsequently, the medium was replaced with FMEM-removed DMEM, and the concentration of 1 nM recombinant myostatin (R & D system, USA) and perilla seaweed ethanol extract (ACEE) by concentration (10.0, 1.0, 0.1, and 0.01 ug / ml) 5%, incubated in a CO ₂ incubator for 24 hours. After 24 hours, the medium was removed and treated with 65 ul of DMEM medium and 65 ul of reagent (Bright-Glo luciferase assay system, USA) to measure luminescence on a microplate luminometer. The measured values were expressed as% of antimyostatin activity using the values of the positive and negative control. Anti myostatin activity was calculated by the following formula.

Myostatin Inhibitory Activity (%) = (Luminescence Value of Experimental Treatments Treated with 1 nM Myostatin Only-Luminous Value of Treatments of Persimmon Seaweed Extracts) X 100 / (Luminous Value of Experimental Treatments Treated with 1 nM Myostatin Only Luminescence Values of the Experimental Zone without Ostatin Treatment)

As a result, it was confirmed that the activity inhibitory effect on myostatin was increased in a concentration-dependent manner when the oleracea ethanol extract was treated, and a trendline formula of y = 11.746 ln (x) + 61.593 was obtained. Based on this trend line, the IC 50 value of the persimmon ethanol extract (ACEE) for 1 nM myostatin was found to be 0.37 ug / ml (FIG. 3A).

<Example 4> Activin A and GDF11 Inhibitory Activity of the Persimmon Seaweed Extract

In order to confirm the inhibitory activity of activin A and GDF11 belonging to a superfamily such as myostatin, the iron extract prepared in Example 1 was used in the same method as the luciferase assay in the antimyostatin activity.

Here, activin A is 0.5 nM, GDF11 was treated with a concentration of 1 nM cells to confirm the Activin A and GDF11 inhibitory activity, was calculated by the following formula.

Anti-Activin A Activity (%) = (Luminescence value of experimental group treated with 0.5nM Activin A only-Luminous value of Molten seaweed extract treated X 100) / (Luminescent value of experimental group treated only with 0.5nM Activin A-Treated with Activin A Luminous Value of Untreated Lab)

Anti-GDF11 activity (%) = (Luminescence value of experimental group treated with 1nM GDF11 only-Luminous value of treated seaweed extract) X 100 / (Luminescent value of experimental group treated with lnM GDF11 only-Luminous value of experimental group not treated with GDF11 )

As a result, the inhibitory activity against 0.5 nM of Activin A and 1 nM of GDF11 in the same superfamily was inhibited by 10, 1, 0.1 ug / ml of ACEE containing the concentration of antimyostatin inhibited. Also did not occur (Fig. 3B, 3C).

In the results, it was confirmed that the oleracea extract does not have toxicity and specifically inhibits myostatin.

Example 5 Myostatin Signal Transduction Inhibition Analysis of Algae Extract

In order to confirm the effect of the horsetail seaweed extract (ACEE) prepared in Example 1 on the myostatin signaling pathway, the degree of phosphorylation of Smad2 transcription factor was confirmed through western blot.

Western blot was performed in the following way. HepG2 cells were seeded at 2.0 × 10 ⁵ cells per well in 6 well plates in DMEM medium (10% FBS, 1% penicillin / streptomycin) and cultured in a 5% CO ₂ incubator. After 24 hours, the medium was replaced with DMEM without FBS, and after 4 hours, 10 nM recombinant myostatin and ACEE extract (perforated seaweed ethanol extract) were treated with IC100 and IC10 concentrations for 30 minutes, respectively (IC100 value of ACEE). Silver 270 ug / ml, IC10 value was 0.125 ug / ml.) After 30 minutes of treatment, cells were washed twice with PBS and then RIPA buffer [20 mM Tris-HCl, pH7.5, 150 mM NaCl, 1 mM Na]. ₂ EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxychloate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM NA ₃ VO ₄ , 1 ug / ml leupeptin (cell signaling, USA)] and protease Inhibitor cocktail and phosphatase inhibitor cocktail (Roche, USA) were treated.

Collected cells were disrupted using a sonicator and centrifuged at 4 ℃, 12,000 rpm for 20 minutes to obtain a supernatant. The obtained supernatant was quantified by BCA assay and electrophoresed on 10% polyacrylamide gel.

After electrophoresis it was transferred to PVDF membrane and blocked for 2 hours at room temperature using 5% BSA or 5% skim milk.

Membrane was washed three times for 10 minutes at room temperature using TBS-T buffer and the primary antibodies [Samd2 (monoclonal antibody), P-Smad2 (monoclonal antibody), β-actin (monoclonal antibody) (Cell signaling, USA)] The reaction was carried out at room temperature for 2 hours.

After washing three times for 10 minutes at room temperature using TBS-T buffer, the secondary antibody [anti-Mouse IgG for β-actin, anti-Rabbit IgG for Smad2 and p-Smad2 (Cell signaling, USA)] at room temperature The reaction was carried out for 2 hours.

After the reaction, Membrane was washed three times for 10 minutes at room temperature using a TBS-T buffer, and was exposed to the x-ray film using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, USA).

As a result, it was confirmed that ACEE inhibited myostatin signal transduction in a concentration-dependent manner through Western blot (inhibition of phosphorylation of Sammd2 transcription factor) in the cell (FIG. 4).

   Specifically, Lane 1 in Figure 4 is a lane that does not process anything to the cell as a control.

Lane 2 is a lane treated only with myostatin, and the signal of myostatin is transferred, so that Smade2 is phosphorylated to increase the expression level of phosphorylated Smad2 (p-Smad2).

Lane 3 is a positive control lane that is simultaneously treated with myostatin and SB431542 (small molecule that inhibits the phosphorylation of the receptor that myostatin binds). The amount of phosphorylated Smad2 expression to be elevated by myostatin (p-smad2) It was confirmed that this does not increase. That is, the signal of myostatin was suppressed by SB431542, and the signal was not transmitted to Smad2, so that phosphorylation did not occur.

Lane 4 is a lane treated with both myostatin and ACEE (IC100). As with lane 3 treated with SB431542, the extract completely inhibited myostatin signal, thereby inhibiting smad2 phosphorylation (p-smad2).

Lane 5 was treated with both myostatin and low concentrations of ACEE (IC10). As a result, it was confirmed that smad2 was phosphorylated (p-smad2) by receiving myostatin. These results indicate that ACEE binds to myostatin in a concentration-dependent manner, preventing myostatin from binding to the receptor, thereby failing to signal Smad2 and inhibiting Smad2 phosphorylation.

Taken together, the results of Luciferase assay and western blot showed that AEB (ACEE) directly inhibited the binding of myostatin and inhibited the phosphorylation of Smad2 by interfering with the receptor of myostatin and not transmitting the signal to Smad2. Confirmed.

<Example 6> Analysis of the effect on the mouse body weight and muscle strength of the horsetail seaweed extract in the animal model

To determine the effect of APO on muscle in vivo, ICR mice were orally administered to confirm the changes in weight and strength.

Specifically, the mice used in the experiment were four-week old male ICR mice orally administered once daily for 21 days. Afterwards, weight and strength tests were conducted on days 0, 7, 14 and 21.

As a result, as a result of oral administration of 21-day ore wakame extract (ACEE) mice weight did not show a significant difference compared to the control (Fig. 5A). However, muscle strength increased 9.93% significantly (p <0.001, p value = 0.000) compared to the control group after oral administration for 14 days, and 8.03% significantly increased compared to the control group after oral administration for 21 days (p <0.001). , p value = 0.000) (Figure 5B).

These results confirmed that oral administration of perilla wakame ethanol extract (ACEE) significantly increased muscle strength without change in mouse weight.

Example 7 Analysis of Muscle Strength Improvement Effect of Algae Fructus Extracts in Animal Models

Cardiopulmonary endurance (cardiopulmonary stamina) or muscle endurance was measured by oral administration to ICR mice as an index for improving muscle mass and muscle strength of the extract of persimmon seaweed extract prepared in Example 1.

Specifically, the mice used in the experiment were four-week old male ICR mice orally administered once daily for 21 days. Then, cardiopulmonary endurance (cardiopulmonary fitness) or muscle endurance test was conducted on days 0, 7, 14, and 21.

Muscular endurance is the ability of a muscle to keep contracting repeatedly for a period of time with respect to resistance, which allows continuous muscle contraction when muscle strength and muscle mass are high. The whole grip test measured the amount of time the mouse hangs while holding the wire net.

Cardiopulmonary endurance (cardiopulmonary fitness) is the ability to perform systemic activities for a long time and can be used as an index for improving muscle mass because it can continuously supply oxygen to the heart and lungs when the muscle mass is high. Cardiopulmonary endurance was measured until exhaustion from the treadmill.

As a result, the whole grip test increased by 110.8% (p <0.1, p value = 0.091) compared to the control group on day 14, and showed a tendency to increase on day 21 but showed no significant difference (FIG. 6A). ).

Treadmill test did not show a significant difference from the control group until the end of oral administration 21 days (Fig. 6B).

Taken together, these results suggest that mice treated with oral seaweed extract (ACEE) significantly increased muscle strength compared to the control group from day 14, so that the same results as Luciferase assay and western blot. By binding to myostatin and inhibiting signal transduction, it was confirmed that the signal of Smad was suppressed and muscle strength increased significantly.

<Example 8> Analysis of the effect on the muscle mass, muscle volume and muscle density of the extract of poultry seaweed in animal model

The muscle weight and volume of the mice tested in Examples 6 and 7 were measured.

Specifically, after oral administration for 21 days, the weight and volume of the front and rear muscles were measured, and muscle density was calculated.

As a result, the weight of the forelimb muscles was significantly increased (p <0.001, p value = 0.000) compared to the control group, and the weight of the hindlimbs muscles was significantly increased compared to the control group, but there was a significant difference. Was absent (FIG. 7A).

The volume of the forelimb muscle was significantly increased by 40.0% (p <0.001, p value = 0.000) compared to the control group, and the hindlimbs muscle volume was significantly increased by 8.61% compared to the control group (p <0.1, p value = 0.065) (FIG. 7B).

Muscle density was not different from the control in both the front and hind legs (FIG. 7C).

Taken together, the Aesculus wakame extract (ACEE) induced significant increase in muscle mass and muscle volume in 21 days. In addition, as in the Luciferase assay, western blot, and muscle increase test results, ACEE extracts were combined with myostatin to inhibit signal transduction, which inhibited Smad signal and significantly increased muscle growth.

Example 9 Analysis of the Effect on the Bone Weight, Bone Volume and Bone Mineral Density

The bone weight and volume of the mice tested in Examples 6 and 7 were measured.

Specifically, after oral administration for 21 days, the weight and volume of the front and rear bones were measured, and bone mineral density was calculated.

As a result, the weight of the forelimb bone was significantly increased by 24.3% (p <0.005, p value = 0.003) compared with the control group, and the hindlimbs bone weight was not significantly different than the control group (Fig. 8A). ).

The volume of the forelimbs and hindlimbs bones tended to increase compared to the control, but there was no significant difference (FIG. 8B).

The bone density was not different from the control group in both the front and hind legs (FIG. 8C).

The results showed that mice fed oral administration of horsetail seaweed extract (ACEE) also affected bone growth, similar to the effect of increasing muscle strength and muscle mass.

<Example 10> Analysis of the effect on the blood of the sea bream seaweed extract in the animal model

Blood of the mice tested in Examples 6 and 7 was analyzed.

Specifically, in order to determine the effect of oral administration of pore seaweed extract on mouse blood, blood samples were collected after oral administration for 21 days, and serum was isolated. Glucose, total cholesterol, and triglyceride levels were analyzed using the separated serum.

As a result, the results of the analysis did not show a significant difference in the case of glucose, total cholesterol and triglyceride (Fig. 9).

In mouse experiments, muscle mass and muscle volume were increased without affecting body weight changes, resulting in an increase in muscle strength and endurance. In blood analysis, glucose, cholesterol and triglycerides in blood were not different from those in the control. In addition, the weight and volume of bones of mice administered oral seaweed extract were found to induce growth. Therefore, it was confirmed that the extract of poultice seaweed affects the growth of bone as well as muscle.

Example 11 Isolation and Purification of Antimyostatin Substances

The antimyostatin material in the oleracea extract was isolated using various solvents.

Specifically, in order to isolate and purify the active substance having antimyostatin activity, the solubility in the organic solvent was confirmed as the first step.

The dried persimmon seaweed powder was obtained using 100% ethanol with light blocked for 24 hours. The obtained extract was removed using a rotary depressurizer to remove the solvent (ethanol), and then fractionated in a separatory funnel by adding a mixture of chloroform and water (1: 1, v: v).

The solvent was removed from the fractionated water fraction layer and the chloroform fraction layer by using a rotary decompression concentrator, and the chloroform fraction layer was added with 90% ethanol and hexane mixture (1: 1, v: v) to separate the funnel. Fraction from In the 90% ethanol fractionation layer and the hexane fractionation layer, the solvent was removed by using a rotary depressurizer, and a mixture of 60% ethanol and chloroform was added to the 90% ethanol fractionation layer (1: 1, v: v). Fractionation in a separatory funnel. The fractionated 60% ethanol fraction layer and the chloroform fraction layer were removed from the solvent using a rotary decompressor.

A total of four fractionation layers (water, hexane, 60% ethanol, chloroform) and crude samples before fractionation were tested for antimyostatin activity at a concentration of 10 ug / ml by luciferase assay system using HEK293 cells.

As a result, it was confirmed that the inhibitory effect of 60% or more against 1 nM myostatin only in the chloroform and 60% ethanol fractionation layers among the four fractionation layers (FIG. 10). In this experiment, it was confirmed that the substance showing the effect of antimyostatin in the extract of pomegranate seaweed is present in the chloroform and 60% ethanol fractionation layer.

Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (17)

Pharmaceutical composition for the prevention and treatment of muscle strengthening or myotropia containing the extract of Agarum cribrosum . The method of claim 1,
The composition has an anti myostatin activity, pharmaceutical composition. The method of claim 1,
The composition improves muscle strength and muscle density or improves bone weight, bone volume and bone density. The method of claim 1,
The composition will have a muscle mass increasing effect, pharmaceutical composition. The method of claim 1,
The extract is a hydrothermal extract or ethanol extract, pharmaceutical composition. The method of claim 1,
Said muscular dystrophy is muscular atrophy, myasthenia gravis, muscular dystrophy, myopathy, muscular dystrophy, muscle weakness, muscle regression axis, muscular dystrophy or myasthenia gravis. Health functional food for the prevention and alleviation of myotropia comprising Agarum cribrosum extract as an active ingredient. Health functional food for strengthening muscle strength, containing Agarum cribrosum extract as an active ingredient. Feed additives containing Agarum cribrosum extract as an active ingredient. The method of claim 9,
The extract is a feed additive which is hot water extract or ethanol extract. (a) extracting by adding a solvent to the dried persimmon seaweed;
(b) concentrating the extract under reduced pressure to remove the solvent; And,
(C) a method for producing a composition for preventing and treating myotropia, comprising recovering the supernatant by centrifuging the poultice seaweed extract obtained in (b). The method of claim 11,
The solvent of step (a) is a method for producing a composition for preventing and treating myotropia, characterized in that water or ethanol. The method of claim 11,
The extraction of step (a) is carried out for 1 to 24 hours, the method for producing a composition for preventing and treating myotropia, characterized in that repeated for 1 to 3 times. The method of claim 11,
The method of preparing a composition for preventing and treating myotropia, characterized in that (a) in the hole seaweed seaweed is contained in a concentration of 10 to 30mg / ml. 15. A pharmaceutical composition for preventing and treating myopathy, comprising an extract of poultice wakame extracted by the method of any one of claims 11-14. A food for the prevention and alleviation of myopathy, comprising an extract of perilla seaweed extracted by the method of any one of claims 11 to 14. A muscle strengthening food comprising the extract of perilla seaweed seaweed extracted by the method of any one of claims 11-14.

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