KR102616724B1 - Composition for improving muscle strength comprising fermented Schisandra chinensis fruit byproduct and method for preparing the same - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating muscle disease containing as an active ingredient a fermented product of Schisandra chinensis fermented by Phellinus linteus (Accession number: KCCM 60261). As a result, while minimizing side effects to the human body, it increases the expression of factors related to muscle cell production, reduces the expression of factors related to muscle cell decomposition, and minimizes muscle cell death, thereby effectively treating various muscle diseases such as muscular dystrophy and sarcopenia and improving muscle strength. can be improved.

Description

Composition for improving muscle strength comprising fermented Schisandra chinensis fruit byproduct and method for preparing the same}

The present invention relates to a composition for improving muscle strength containing fermented Schisandra chinensis as an active ingredient and a method for producing the same. More specifically, it relates to a composition containing fermented Schisandra chinensis as an active ingredient and a method for producing the same.

Muscle atrophy refers to a phenomenon in which muscle function deteriorates and mass decreases due to excessive protein breakdown. Skeletal muscle is called a voluntary muscle, and it not only plays a functional role in maintaining our body's posture and promoting movement, but also plays a major role in whole body homeostasis, including participating in glucose metabolism. In Korea, the proportion of the population aged 60 or older is expected to more than double by 2050, and the increase in the elderly population worldwide is emerging as an important social problem. This increase in the elderly population is associated with an increase in the rate of a disease called muscular atrophy. It is also closely related to Muscle atrophy has multiple etiologies, including loss of muscle mass, weakness, fatigue, and decreased muscle contractile activity, providing an aggravating etiological factor in chronic diseases such as chronic heart failure, chronic kidney disease, obstructive pulmonary disease, and cancer. Atrophy is a major factor affecting quality of life and increased mortality and morbidity. Recently, muscle atrophy has been known to be one of the symptoms that cause sarcopenia, and accordingly, there is a need to develop specialized treatments, including drug treatment, for muscular atrophy, which is attracting attention from researchers as a research topic around the world. .

It is already well known that endogenous glucocorticoid (GC) is a stress hormone that plays an important role in regulating energy homeostasis in mammals. On the other hand, excessive GC stimulation is also associated with side effects such as hyperglycemia, insulin resistance, fatty liver, muscle atrophy, and severe metabolic dysfunction. Dexamethasone (Dex), a synthetic GC, is a medicine commonly used to treat various inflammatory diseases, including rheumatoid arthritis, bronchial asthma, and systemic lupus erythematosus. When administered in high doses or for long periods of time, it has serious side effects, including loss of muscle mass and functional impairment. It can cause it. Excessive dexamethasone induces muscle atrophy by inhibiting glucose consumption and utilization due to impaired insulin signaling, and is therefore used as a drug to induce muscle atrophy in both in vivo and in vitro studies. In particular, activation of proteolytic factors by activation of muscle-specific Atrogin-1 and MuRF-1, an E3 ubiquitin ligase, has been reported in a dexamethasone-induced muscle atrophy model.

In Oriental medicine, diseases including muscular atrophy, myasthenia gravis, muscular dystrophy, muscular atrophy, and motor neuron disease are included in the wilt disease, which causes overall weakness in body functions, including muscle deterioration, making recovery of function increasingly difficult. It refers to a disease that causes muscle atrophy along with limb deformity due to weakness or inactivity of the muscles and veins of the limbs for a long period of time. Therefore, in Oriental medicine, muscle atrophy is included in the category of symptoms rather than diseases, and these symptoms represent four major conditions. First, irregular eating damages the spleen and stomach, causing symptoms that affect qi and blood transformation deficiency; second, symptoms that affect mood swings, blood storage in the liver, and physical activity ability due to liver dryness; third, heat and heat The energy, blood, and body fluids included were classified into symptoms of body fluid damage due to internal organs, intestines, limbs, muscles, and tendons, and fourthly, symptoms of loss of kidney function. Therefore, traditional medicine believes that muscle atrophy is mainly caused by liver and kidney deficiency, and weakness of the spleen and stomach. Ultimately, muscle atrophy refers to a decline in physical function caused by an imbalance in the body's homeostasis.

Over the past few decades, people have attempted to research and develop the molecular mechanisms of muscle atrophy, but effective drugs or treatments have not yet been developed. Currently, the main treatments include exercise training, nutritional supplements, and physical therapy rather than pharmacological treatment. However, exercise therapy is severely limited in the elderly, bedridden population, and those with acute illnesses, and although many nutrients have been shown to be beneficial for muscle wasting, most are still limited to patients with primary muscle wasting. am.

Several drugs or functional substances to improve muscle strength are being studied to treat muscle atrophy, but few have been clinically approved, and there is a need to develop effective muscular atrophy treatments or supplements with fewer side effects.

Korean Patent Publication No. 10-1616170

The purpose of the present invention is to solve the above problems by increasing the expression of factors related to muscle cell production, reducing the expression of factors related to muscle cell decomposition, and minimizing muscle cell death while minimizing side effects on the human body, thereby preventing muscular dystrophy and sarcopenia. The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating muscle diseases that can effectively treat various muscle diseases, and a method for manufacturing the same.

Another purpose of the present invention is to solve the above problems by increasing the expression of factors related to muscle cell production while minimizing side effects on the human body, reducing the expression of factors related to muscle cell decomposition, and increasing muscle mass by minimizing muscle cell death. The aim is to provide a food composition for improving muscle strength and a manufacturing method thereof that can minimize muscle loss.

Another purpose of the present invention is to solve the above problems by increasing the expression of factors related to muscle cell production, reducing the expression of factors related to muscle cell decomposition, and minimizing muscle cell death while minimizing side effects on the human body, thereby increasing muscle mass. The goal is to provide health functional foods for improving muscle strength that can increase muscle strength and minimize muscle loss, and a manufacturing method thereof.

According to one aspect of the present invention,

A pharmaceutical composition for preventing or treating muscle disease is provided, comprising as an active ingredient a fermented product of Schisandra chinensis fermented by Phellinus linteus (Accession number: KCCM 60261).

The fermented product of Schisandra chinensis is a solid-state fermented product of Schisandra chinensis, and the solid-state fermented product of Schisandra chinensis may be solid-state fermented in a state where the moisture content of Schisandra chinensis powder is 45 to 60 wt%.

The moisture of Schisandra chinensis powder during the solid-state fermentation may be deep ocean water with a hardness of 80 to 250 ppm.

The solid-state fermented product of Schisandra chinensis may be an extract of the solid-state fermented product of Schisandra chinensis bark extracted with an extraction solvent.

The extraction solvent is selected from water, lower alcohols having 1-4 carbon atoms, mixed solvents of the lower alcohols and water, acetone, ethyl acetate, chloroform, butyl acetate, 1,3-butylene glycol, hexane, and diethyl ether. It could be any one.

The muscle disease is any one selected from muscular atrophy, sarcopenia, atony, muscular dystrophy, myasthenia gravis, and amyotrophic lateral sclerosis. You can.

The pharmaceutical composition for preventing or treating muscle disease may be used to increase the expression of one or more types of muscle cell production-related factors selected from Myo-D, Myogenin, MEF2, Myf5, Myf6, and pAMPK.

The pharmaceutical composition for preventing or treating muscle disease may be used to inhibit the expression of one or more types selected from Atrogin-1, MuRF-1, FoxO3α, and Myostatin, which are factors related to muscle cell breakdown.

According to another aspect of the present invention,

(a) drying and pulverizing Schisandra chinensis to prepare Schisandra chinensis powder;

(b) mixing the Schisandra chinensis powder with an aqueous solution of sugars and then sterilizing it to prepare a sterilized Schisandra chinensis pumpkin mixture; and

(c) inoculating the sterilized Schisandra chinensis mixture with Phellinus linteus , KCCM 60261 and performing solid-state fermentation to produce a solid-state fermented Schisandra chinensis product; a method for producing a pharmaceutical composition for preventing or treating muscle disease comprising: provided.

In step (b), the mixing of the saccharide aqueous solution can cause the moisture content of the sterilized Schisandra chinensis cucumber mixture to be 45 to 60 wt%.

In step (b), the aqueous saccharide solution may include deep ocean water with a hardness of 80 to 250 ppm.

In step (c), the Sanghwang mushroom fungus can be inoculated at 2 to 10 parts by weight based on 100 parts by weight of the sterilized Schisandra chinensis mixture.

In step (c), the solid-state fermentation may be performed at 20 to 28°C for 30 to 40 days.

After step (c), (d) mixing the Schisandra chinensis solid-state fermented product with an extraction solvent, extracting it, and filtering it to prepare a solid-state fermented extract of Schisandra chinensis can be further performed.

According to another aspect of the present invention,

A food composition for improving muscle strength is provided, comprising as an active ingredient a fermented product of Schisandra chinensis fermented by Phellinus linteus (Accession number: KCCM 60261).

The fermented product of Schisandra chinensis is a solid-state fermented product of Schisandra chinensis, and the solid-state fermented product of Schisandra chinensis may be solid-state fermented in a state where the moisture content of Schisandra chinensis powder is 45 to 60 wt%.

The food composition for improving muscle strength may be used to increase muscle mass or inhibit muscle loss.

According to another aspect of the present invention,

(a) drying and pulverizing Schisandra chinensis to prepare Schisandra chinensis powder;

(b) mixing the Schisandra chinensis powder with an aqueous solution of sugars and then sterilizing it to prepare a sterilized Schisandra chinensis pumpkin mixture; and

(c) inoculating the sterilized Schisandra chinensis mixture with Phellinus linteus , KCCM 60261 and performing solid-state fermentation to produce a solid-state fermented Schisandra chinensis plant. A method for producing a food composition for improving muscle strength is provided, including the step of:

According to another aspect of the present invention,

A health functional food for improving muscle strength containing the food composition for improving muscle strength is provided.

According to another aspect of the present invention,

(a) drying and pulverizing Schisandra chinensis to prepare Schisandra chinensis powder;

(b) mixing the Schisandra chinensis powder with an aqueous solution of sugars and then sterilizing it to prepare a sterilized Schisandra chinensis pumpkin mixture; and

(c) inoculating the sterilized Schisandra chinensis mixture with Phellinus linteus , KCCM 60261 and subjecting it to solid-state fermentation to produce a Schisandra chinensis solid-state fermented product. A method for manufacturing a health functional food for improving muscle strength is provided, including: .

The pharmaceutical composition for preventing or treating muscle diseases of the present invention minimizes side effects on the human body while increasing the expression of factors related to muscle cell production, reducing the expression of factors related to muscle cell decomposition, and minimizing muscle cell death, thereby preventing muscular dystrophy and sarcopenia. It can effectively treat various muscle diseases.

The food composition for improving muscle strength of the present invention increases the expression of factors related to muscle cell production, reduces the expression of factors related to muscle cell decomposition, and minimizes muscle cell death, while minimizing side effects on the human body, thereby increasing muscle mass and preventing muscle loss. It can be minimized.

The health functional food for improving muscle strength of the present invention increases the expression of factors related to muscle cell production, reduces the expression of factors related to muscle cell decomposition, and minimizes muscle cell death, while minimizing side effects on the human body, thereby increasing muscle mass and reducing muscle loss. can be minimized.

Figure 1 is a micrograph of the degree of differentiation of myotube cells according to the cell culture period in Experimental Example 1.
Figure 2 shows the results of measuring the length (lengh) (a) and thickness (b) of myotube cells according to the cell culture period according to Experimental Example 1.
Figure 3 shows a micrograph (a) and the length (lengh) and width (b) of myotube cells as a result of treatment with atorvastatin at different concentrations after differentiation of muscle cells for 6 days.
Figure 4 shows the micrograph (a) and the length (lengh) and width (b) of myotube cells as a result of measuring the width and length of myotube cells treated with atorvastatin at a concentration of 10 μM for 24 hours according to Experimental Example 1. It is shown.
Figure 5 shows the micrograph (a) and the length (lengh) and width (b) of myotube cells as a result of measuring the width and length of myotube cells treated with atorvastatin at a concentration of 10 μM for 48 hours according to Experimental Example 1. It is shown.
Figures 6 and 7 show the cytotoxicity evaluation results according to Experimental Example 2.
Figure 8 is a microscope image for verifying the effect of improving muscular dystrophy according to cell shape analysis in Experimental Example 3.
Figure 9 shows the results of RT-qPCR analysis for analyzing the expression of muscle cell-generating transcription factors according to the type of solid-phase fermentation extract in Experimental Example 4.
Figure 10 shows the results of RT-qPCR analysis for analyzing the expression of muscle cell production transcription factors according to the treatment concentration of the solid-phase fermentation extract of Example 1 of Experimental Example 4.
Figure 11 shows the results of RT-qPCR analysis for analysis of the expression of muscle cell degradative transcription factors according to the treatment concentration of the solid-phase fermentation extract of Example 1 of Experimental Example 4.
Figure 12 shows the degree of ROS and glutathione production according to Experimental Example 5.
Figure 13 shows the results of RT-qPCR analysis for apoptosis regulators according to Experimental Example 6.
Figure 14 shows the results of Western blot analysis for apoptosis-related proteins according to Experimental Example 6.
Figure 15 is a schematic diagram of the animal experiment design.
Figure 16 shows the results of analysis of body weight change in the dexamethasone-induced muscular atrophy animal model of Experimental Example 7.
Figure 17 shows the grip test measurement results of Experimental Example 8.
Figure 18 shows the mouse running test (Treadmill test) results of Experimental Example 8.
Figure 19 is a photograph of the femoral muscle removed in Experimental Example 9.
Figure 20 shows the muscle thickness and weight measurement results for measuring the quadriceps muscle protection effect in Experimental Example 9.
Figure 21 is an optical microscope image stained with H&E for analysis of morphological changes in the muscle of Experimental Example 10.
Figure 22 shows the results of analysis of the effect on mRNA expression related to muscle breakdown/production in Experimental Example 11.

Since the present invention can be modified in various ways and can have various embodiments, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all transformations, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

The pharmaceutical composition for preventing or treating muscle disease of the present invention contains as an active ingredient a fermented product of Schisandra chinensis fermented by Phellinus linteus (Accession number: KCCM 60261).

The fermented product of Schisandra chinensis is a solid-state fermented product of Schisandra chinensis, and the solid-state fermented product of Schisandra chinensis is preferably solid-state fermented with the moisture content of the Schisandra chinensis powder being 45 to 60 wt%, more preferably 50 to 55 wt%. It may have been solid-state fermented. At this moisture content, the activity of Sanghwang mushroom bacteria is high, so fermentation can be carried out efficiently.

Solid state fermentation (SSF) has lower facility construction costs than liquid fermentation methods and can efficiently mass-produce fermented products without special equipment, so it can be widely used in the fermentation industry where production facilities are inexpensive.

The solid-state fermented product of Schisandra chinensis may be an extract of the solid-state fermented product of Schisandra chinensis bark extracted with an extraction solvent.

The extraction solvent is selected from water, lower alcohols having 1-4 carbon atoms, mixed solvents of the lower alcohols and water, acetone, ethyl acetate, chloroform, butyl acetate, 1,3-butylene glycol, hexane, and diethyl ether. Any one may be used, preferably water or a lower alcohol having 1 to 4 carbon atoms, more preferably water.

The muscle disease is any one selected from muscular atrophy, sarcopenia, atony, muscular dystrophy, myasthenia gravis, and amyotrophic lateral sclerosis. It may be muscular dystrophy or sarcopenia.

The pharmaceutical composition for preventing or treating muscle disease may be used to increase the expression of one or more types of muscle cell production-related factors selected from Myo-D, Myogenin, MEF2, Myf5, and Myf6.

The pharmaceutical composition for preventing or treating muscle disease may be used to inhibit the expression of one or more types selected from Atrogin-1, MuRF-1, FoxO3α, and Myostatin, which are factors related to muscle cell breakdown.

Hereinafter, the method for producing the pharmaceutical composition for preventing or treating muscle disease according to the present invention will be described.

First, Schisandra chinensis powder is prepared by drying and pulverizing Schisandra chinensis fruit (step a).

Next, the Schisandra chinensis powder is mixed with an aqueous solution of sugars and then sterilized to prepare a sterilized Schisandra chinensis pumpkin mixture (step b).

The sugars may be sugar, glucose, fructose, etc., and sugar may be preferably used.

The aqueous concentration of sugars is preferably 3 to 10%, more preferably 5 to 7%.

The mixing of the saccharide aqueous solution is preferably such that the moisture content of the sterilized Schisandra chinensis pumpkin mixture is 45 to 60 wt%, and more preferably 50 to 55 wt%. Within the above range, the activity of Sanghwang mushroom bacteria is high and solid-state fermentation can be performed efficiently.

The aqueous sugar solution is preferably prepared using deep ocean water with a hardness of 80 to 250 ppm, and more preferably, deep ocean water with a hardness of 150 to 220 ppm can be used. When such deep sea water is used, the beta-glucan content of the final fermentation product can be increased. If the hardness is lower than 80, the beta-glucan content decreases, and if it exceeds 250 ppm, the activity of Phellodendron mushrooms may decrease.

Next, Phellinus linteus , KCCM 60261 is inoculated into the sterilized Schisandra chinensis mixture and subjected to solid-state fermentation to prepare a solid-state fermented Schisandra chinensis product (step c).

The Sanghwang mushroom fungus can be inoculated in an amount of 2 to 10 parts by weight, preferably in an amount of 3 to 6 parts by weight, based on 100 parts by weight of the sterilized Schisandra chinensis cucumber mixture. The muscle-improving activity of the solid-phase fermented product of Schisandra chinensis produced at this inoculation concentration appears, and if it is outside the above range, the muscle-improving activity appears at a level similar to that of unfermented Schisandra chinensis, and the efficacy may be significantly reduced.

The solid-state fermentation is preferably performed at 20 to 28°C for 30 to 40 days, and more preferably at 22 to 26°C for 33 to 37 days. Under these solid-state fermentation conditions, the activity of Sanghwang mushroom bacteria is excellent.

Thereafter, the solid-state fermented Schisandra chinensis extract is mixed with an extraction solvent, extracted, and filtered to prepare a solid-state fermented Schisandra chinensis extract (step d).

The extraction solvent is selected from water, lower alcohols having 1-4 carbon atoms, mixed solvents of the lower alcohols and water, acetone, ethyl acetate, chloroform, butyl acetate, 1,3-butylene glycol, hexane, and diethyl ether. Any one may be used, preferably water or a lower alcohol having 1 to 4 carbon atoms, more preferably water.

It is preferable to freeze-dry the solid fermented product of Schisandra chinensis and then pulverize it to use it in powder form.

When the extraction solvent is water, it is preferable to extract using hot water at 90 to 100°C.

The term ‘extract’ used in this specification has a meaning commonly used in the art as a crude extract, but in a broad sense, it may also include fractions obtained by additional fractionation of the extract. Additionally, it can be produced in powder form by additional processes such as reduced pressure distillation and freeze drying or spray drying.

Meanwhile, in this specification, the term ‘including as an active ingredient’ means containing a sufficient amount to achieve the efficacy or activity of the fermented product of Schisandra chinensis or its extract. In one embodiment of the present invention, the fermented product of Schisandra chinensis in the composition of the present invention is, for example, 0.001 mg/kg or more, preferably 0.1 mg/kg or more, more preferably 10 mg/kg or more, and even more. Preferably it contains 100 mg/kg or more, even more preferably 250 mg/kg or more, and most preferably 0.1 g/kg or more. Since the fermented product of Schisandra chinensis or its extract is a natural product and has no side effects on the human body even when administered in excessive amounts, the upper quantitative limit of Schisandra chinensis contained in the composition of the present invention can be selected within an appropriate range by a person skilled in the art.

The pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable auxiliaries in addition to the active ingredients, and the auxiliaries include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, and glidants. Alternatively, flavoring agents, etc. may be used.

For administration, the pharmaceutical composition may be preferably formulated as a pharmaceutical composition containing one or more pharmaceutically acceptable carriers in addition to the active ingredients described above.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an orally, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacance or sodium oleate, sodium stearate, magnesium stearate, sodium Includes benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, etc.

Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and these. One or more of the ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., and is preferably administered orally.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and is usually A skilled doctor can easily determine and prescribe an effective dosage for desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g/kg.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using pharmaceutically acceptable carriers and/or excipients according to a method that can be easily performed by those skilled in the art. It can be manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, or capsule, and may additionally contain a dispersant or stabilizer.

In addition, the present invention provides a food composition for improving muscle strength containing as an active ingredient a fermented product of Schisandra chinensis fermented by Phellinus linteus (Accession number: KCCM 60261).

The fermented product of Schisandra chinensis is a solid-state fermented product of Schisandra chinensis, and the solid-state fermented product of Schisandra chinensis may be solid-state fermented in a state where the moisture content of Schisandra chinensis powder is 45 to 60 wt%.

The description of the Schisandra chinensis fermented product fermented by Phellinus linteus (Accession number: KCCM 60261) and its extract are the same as those described in the pharmaceutical composition, so please refer to the above for details.

Additionally, the present invention provides a method for producing a food composition for improving muscle strength.

Since the manufacturing method of the food composition for improving muscle strength of the present invention is the same as the manufacturing method of the pharmaceutical composition for preventing or treating muscle disease described above, refer to that section for specific details.

The food composition according to the present invention can be used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, bread, ramen, other noodles, gum, ice cream, vitamin complexes, and health supplements. There are food items, etc.

The food composition of the present invention may include not only fermented Schisandra chinensis or extracts thereof as active ingredients, but also ingredients commonly added during food production, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavors. Includes provisions. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is manufactured as a drink or beverage, in addition to the fermented product of Schisandra chinensis or its extract of the present invention, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts are added. Can be included.

The present invention provides a health functional food containing a food composition containing fermented Schisandra chinensis or an extract thereof as an active ingredient, and a method for producing the same. Health functional foods are foods manufactured by adding fermented Schisandra chinensis or its extracts to food ingredients such as beverages, teas, spices, gums, and confectionery, or by encapsulating, powdering, or suspending them, and consuming them may cause certain health problems. It means that it is effective, but unlike regular drugs, it has the advantage of not having any side effects that may occur when taking the drug for a long time because it is made from food. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of fermented Schisandra chinensis or extract thereof in such health functional foods cannot be uniformly specified as it varies depending on the type of health functional food being targeted. However, it may be added within the range that does not damage the original taste of the food. It is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, relative to food. In addition, in the case of health functional foods in the form of pills, granules, tablets, or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule, or beverage.

Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are merely illustrative of the present invention, and it is clear to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention. It is natural that such variations and modifications fall within the scope of the attached patent claims.

[Example]

Example 1: Schisandra chinensis solid-state fermentation extract using Sanghwang mushroom fungi

(1) Phellodendron agaris ( Phellinus linteus ) (KCCM 60261)) solid-state fermentation powder production

After producing the Schisandra chinensis extract, the residue remaining, Schisandra chinensis, was dried with hot air at 60°C for 48 hours, and the dried Schisandra chinensis was pulverized using a grinder. The crushed Schisandra chinensis was mixed with 5% sugar water in a solid-state fermentation vessel in a ratio of 50:50 with Schisandra chinensis, sterilized at 121℃ 1.1kg/cm ² for 30 minutes, and then sterilized at 5℃/min for 25 minutes. Cooled to ℃. It was prepared by inoculating 5% of Phellinus linteus (KCCM 60261) bacteria into cooled Schisandra chinensis gourd and performing solid-state fermentation in an incubator at 24 ± 2°C for 50 days. Accordingly, the solid-state fermented product of Schisandra chinensis produced was sterilized at 121°C and 1.1kg/cm ² for 30 minutes, and the solid-state fermented product with all bacteria killed was freeze-dried at -40°C and pulverized for 1 minute with a grinder (grinder power 3000W). Schisandra chinensis solid fermented powder was obtained.

(2) Manufacture of Schisandra chinensis bark solid-state fermentation extract

As described above, 100 g of Schisandra chinensis solid phase fermentation powder prepared using Sanghwang mushroom fungus was mixed with 900 g of distilled water, then subjected to hot water extraction at 100°C for 2.5 hours. The extracted liquid was filtered through a 100 mm paper filter, and the filtrate was filtered again at 0.45 ㎛. After that, it was freeze-dried at 40°C to obtain a solid-state fermented extract of Schisandra chinensis.

Example 2: Schisandra chinensis solid phase fermentation extract using Sanghwang mushroom fungus and deep ocean water

A solid-state fermentation extract of Schisandra chinensis was prepared under the same conditions as in Example 1, except that deep sea water with a hardness of 100 ppm was used instead of water when preparing 5% concentration sugar water.

Example 3: Schisandra chinensis solid phase fermentation extract using Sanghwang mushroom fungus and deep ocean water

A solid-state fermentation extract of Schisandra chinensis was prepared under the same conditions as in Example 1, except that deep sea water with a hardness of 200 ppm was used instead of water when preparing 5% concentration sugar water.

Comparative Example 1: Preparation of Schisandra chinensis solid-state fermentation extract using Cordyceps sinensis

A solid-state fermentation extract of Schisandra chinensis was prepared under the same conditions as in Example 1, except that Cordyceps militaris (KCCM60304) was used instead of the fungus S. chinensis.

Comparative Example 2: Preparation of Schisandra chinensis solid-state fermentation extract using shiitake mushrooms

A solid-state fermentation extract of Schisandra chinensis was prepared under the same conditions as in Example 1, except that shiitake mushrooms were used instead of Sanghwang mushrooms.

Comparative Example 3: Preparation of Schisandra chinensis solid-state fermentation extract using Reishi mushroom fungus

A solid-state fermentation extract of Schisandra chinensis was prepared under the same conditions as in Example 1, except that Ganoderma lucidum was used instead of the Sage mushroom fungus.

Comparative Example 4: Preparation of Schisandra chinensis root fermentation extract using Ganoderma lucidum fungus

A solid-state fermentation extract of Schisandra chinensis was prepared under the same conditions as in Example 1, except that natto bacteria were used instead of Sanghwa mushroom bacteria.

Comparative Example 5: Preparation of Schisandra chinensis solid-state fermentation extract using Reishi mushroom fungus

A solid-state fermentation extract of Schisandra chinensis was prepared under the same conditions as in Example 1, except that Lactobacillus plantarum was used instead of Sanghwa mushroom bacteria.

Comparative Example A: Schisandra chinensis solid-state fermentation extract using Sanghwang mushroom bacteria

A solid-state fermentation extract of Schisandra chinensis was prepared under the same conditions as Example 1, except that Schisandra chinensis, not Schisandra chinensis, was used as the material.

Comparative Example B: Schisandra chinensis solid-state fermentation extract using Cordyceps sinensis

A solid-state fermentation extract of Schisandra chinensis was prepared under the same conditions as Comparative Example 1, except that Schisandra chinensis, not Schisandra chinensis, was used as the material.

Comparative Example C: Schisandra chinensis solid-state fermentation extract using shiitake mushrooms

A solid-state fermentation extract of Schisandra chinensis was prepared under the same conditions as Comparative Example 2, except that Schisandra chinensis instead of Schisandra chinensis was used as the material.

Comparative Example D: Schisandra chinensis solid-state fermentation extract using Reishi mushroom

A solid-state fermentation extract of Schisandra chinensis was prepared under the same conditions as Comparative Example 3, except that Schisandra chinensis instead of Schisandra chinensis was used as the material.

Comparative Example E: Schisandra chinensis solid-state fermentation extract using natto bacteria

A solid-state fermentation extract of Schisandra chinensis was prepared under the same conditions as Comparative Example 4, except that Schisandra chinensis, not Schisandra chinensis, was used as the material.

Comparative Example E: Schisandra chinensis solid-state fermentation extract using lactic acid bacteria

A solid-state fermentation extract of Schisandra chinensis was prepared under the same conditions as Comparative Example 5, except that Schisandra chinensis instead of Schisandra chinensis was used as the material.

Beta-glucan content analysis

Table 1 below shows the results of analyzing the beta-glucan content of Schisandra chinensis solid-state fermented extracts prepared by Comparative Example A and Examples 1, 2, and 3, respectively.

division ingredient Moisture content during solid-state fermentation Beta-glucan content (wt%) Comparative example A Schisandra water 5.08 Example 1 Schisandra Pak water 9.95 Example 2 Schisandra Pak Deep ocean water hardness 100ppm 10.87 Example 3 Schisandra Pak Deep ocean water hardness 200ppm 12.34

According to this, the beta-glucan content was higher when Schisandra chinensis was used as an ingredient than when Schisandra chinensis was used as an ingredient. In addition, the beta-glucan content was found to be higher when deep sea water was used during Schisandra chinensis fermentation than when water was used. In particular, the highest beta-glucan content was measured when deep sea water with a hardness of 200ppm was used.

[Experimental example: Intracellular experiment]

Experimental Example 1: Muscle cell culture and muscle loss induction

Myoblast C2C12 was cultured in DMEM (Dulbecco's Modified Eagle's) medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C and 5% CO ₂ conditions. After C2C12 cells were sufficiently proliferated in a 75 cm ² flask, they were cultured for 6 days in DMEM medium supplemented with 2% horse serum and 1% penicillin to induce differentiation into myotube cells. They were induced by replacement at daily intervals, and micrographs of the degree of differentiation of myotube cells according to the cell culture period are shown in Figure 1, and the results of measuring myotube cell length (lengh) (a) and thickness (b) are shown in Figure 1. It is shown in Figure 2. According to this, the formation of myotube cells was observed on days 0, 2, 4, 6, and 8 after the start of differentiation, and the degree of differentiation was determined by observing the length and width of myotube cells. As a result, the differentiation of muscle cells showed that the length of the muscle was the longest on the 6th and 8th days, and the width of the muscle was largest on the 6th day and decreased slightly from the 8th day. Therefore, muscle loss differentiation conditions were established on the 6th day after differentiation induction, and subsequent experiments were conducted.

Meanwhile, in order to establish a model for muscle loss due to differentiation of muscle cells, muscle loss was induced with Atorvastatin, a statin drug known to cause muscle loss through protein decomposition of muscle cells. First, muscle cells were differentiated for 6 days, and then treated with atorvastatin at different concentrations of 5, 10, and 20 μM. The micrograph (a) and myotube cell length (lengh) and width (b) are shown in Figure 3. indicated. According to this, it was confirmed that the width and length of myotube cells decreased in an atorvastatin concentration-dependent manner.

In addition, the width and length of myotube cells treated with atorvastatin at a concentration of 10 μM for 24 hours were measured, and the micrograph (a) and myotube cell length (lengh) and width (b) are shown in Figure 4. The micrograph (a) and the length (lengh) and width (b) of myotube cells upon treatment for 48 hours are shown in Figure 5. According to this, there was little difference between the group treated with 10 μM atorvastatin for 24 hours and the group treated for 48 hours. Therefore, on the 6th day of differentiation of C2C12 cells, muscle loss was induced by treating C2C12 cells with 10 μM of atorvastatin and culturing them for 24 hours to construct a muscle loss model.

Experimental Example 2: Cytotoxicity evaluation

To determine cytotoxicity, the MTT assay method was used. The cultured C2C12 cells were distributed in 200 ㎕ each at a concentration of 1 × 10 ⁵ cell/㎖ in a 96-well plate and cultured for 24 hours. C2C12 cells were treated with the solid-phase fermentation extract samples of Example 1, Comparative Examples 1 to 5, and Comparative Examples A to F at concentrations of 0.25, 0.5, 1.0, 2.5, 5.0, and 10 mg/ml, and then reacted for 24 hours, A solution of 5 mg/mL (DPBS) MTT reagent diluted 5 times in medium was dispensed at 100 ㎕/well and reacted in an incubator for 1 hour. After removing the supernatant, formazan was dissolved by treating with 100 μl of DMSO, and then the absorbance was measured at 570 nm using a micro reader, and the results are shown in Figures 6 and 7.

According to this, most of the toxicity was shown at the highest concentration of 10 mg/ml in all experimental groups. In general, when treating cells with the extract, considering the highest concentration of 1.0 mg/ml, the cell survival rate of C2C12 myotube cells was over 90%, so it was judged that there was almost no cytotoxicity. Therefore, in subsequent experiments, samples were used up to 200 μg/ml.

Experimental Example 3: Evaluation of the effect of improving muscular dystrophy according to cell shape analysis

Differentiated C2C12 myotube cells were treated with the Schisandra chinensis solid-phase fermentation extract of Example 1 and the Schisandra chinensis solid-state fermentation extract of Comparative Example A at a concentration of 200 ㎍/㎖, and the results of microscopic observation of the cells were examined to determine their efficacy against muscle atrophy. It is shown in Figure 8. According to this, the group treated with the solid-state fermentation extract of Example 1, which was subjected to solid-state fermentation under the same conditions using Sanghwa mushrooms but using Schisandra chinensis as an ingredient, was found to have an effect on improving muscle atrophy, but the group treated with the solid-state fermentation extract of Comparative Example A It was found that there was little effect on improving muscle atrophy.

Experimental Example 4: Expression analysis of factors related to muscle cell production or degradation

Example 1, Comparative Examples 1 to 5, and Comparative Examples A to F were treated with the solid-phase fermentation extracts of Comparative Examples A to F at a concentration of 200 ㎍/㎖ to induce muscle atrophy to induce myogenin and Myo-, which are related to muscle cell generation transcription factors. Quantitative RT-PCR analysis was performed to analyze the expression of D mRNA.

After inducing muscle atrophy as described above, the medium was removed, washed with PBS, and RNA was extracted using the RNeasy® mini kit (Aiagen, Hilden, Gerbany). The extracted RNA was quantified using a spectrophotometer (Nanodrop), and 1 μg RNA was synthesized into cDNA using the Maxima frist strand cDNA synthesis kit for RT-qPCR. 40 PCR cycles were performed using 10 ㎕ of PCR bio syGreen blue Mix (PCR Biosystems, Pennsylvania, USA), 19 ㎕ of a mixture containing 2 ㎕ of primer, and 1 ㎕ of cDNA. Information on primers used in the polymerase reaction is summarized in Table 2 below.

Target Primer Sequences MyoD Forward 5’-GATGGCATGATGGATTACAG-3’ Reverse 5’-CTCCACTATGCTGGACAGG-3’ Myogenin Forward 5’-AGTACATTGAGCGCCTACAG-3’ Reverse 5’-GACGTAAGGGAGTGCAGATT-3’ Atrogin-1 Forward 5’-CTGCCTTGTGCTTACAACT-3’ Reverse 5’-TGCTCTCTTCTTGGGTAACA-3’ Myf5 Forward 5’-TGAGGGAACAGGTGGAGAAC-3’ Reverse 5’-AGCTGGACACGGAGCTTTTA-3’ Myf6 Forward 5’-ATTCTTGGCGGGTGCGGATTT-3’ Reverse 5’-ACGTTTGCTCCTCCTTCCTT-3’ MEF2 Forward 5’-TCCATCAGCCATTTCAACAA-3’ Reverse 5’-GTTACAGAGCCGAGGTGGAG-3’ FoxO3 Forward 5’-ACAAACGGCTCACTTTGTCC-3’ Reverse 5’-GTGCCGGATGGAGTTCTTC-3’ Myostatin Forward 5’-CTGTAACCTTCCCAGGACCA-3’ Reverse 5’-GCAGTCAAGCCCAAAGTCTC-3’ MuRF1 Forward 5’-TGCCTACTTGCTCCTTGTGC-3’ Reverse 5’-CACCAGCATGGAGATGCAGT-3’

The results of RT-qPCR analysis comparing the expression levels of myogenin (a) and Myo-D (b) for Example 1, Comparative Examples 1 to 5, and Comparative Examples A to F are shown in Figure 9. (1: Comparative Example A, 2: Comparative Example B, 3: Comparative Example C, 4: Comparative Example D, 5: Comparative Example E, 6: Comparative Example F, 7: Examples 1, 8: Comparative Examples 1, 9 : Comparative Examples 2, 10: Comparative Examples 3, 11: Comparative Examples 4, 12: Comparative Example 5). According to this, it can be confirmed that the expression of myogenin and Myo-D, which are related to muscle cell generation transcription factors, was significantly higher in the group treated with the Schisandra chinensis solid phase fermentation extract of Example 1 compared to the groups treated in other comparative examples. In other words, it can be seen that the solid-phase fermentation extract of Schisandra chinensis by the Sanghwa mushroom fungus of the present invention has the effect of improving muscle atrophy by promoting the production of muscle cells. Meanwhile, RT-qPCR analysis was performed to analyze the expression level of factors related to muscle cell production and degradation related to the treatment of Schisandra chinensis bark solid-phase fermentation extract by Sanghwang mushroom of Example 1 at different concentrations of 50, 100, and 200 ㎍/㎖. was carried out.

After analysis, the results of RT-qPCR analysis for Myo-D, Myogenin, MEF2, Myf5, and Myf6, which are factors related to muscle cell generation, are shown in Figure 10. According to this, it can be seen that above the concentration of 100 μg/ml, the expression amount increases in a concentration-dependent manner.

In addition, the results of RT-qPCR analysis for Atrogin-1, MuRF-1, FoxO3α, and Myostatin, which are factors related to muscle cell breakdown, are shown in Figure 11. According to this, it was confirmed that the expression level of all factors was increased by atorvastatin treatment, and it was confirmed that the mRNA expression level was reduced in a concentration-dependent manner when treated with Schisandra chinensis solid phase fermented extract of Example 1 ( ^** p < 0.01 and ^*** p<0.001 vs. CTL; ^# p<0.05, ^## p<0.01 and ^### p<0.001 vs. ATV treated group).

Experimental Example 5: Glutathione (GSH) and ROS production analysis

Sarcopenia, a condition in which muscle strength is weakened, generates reactive oxygen species in muscle cells, which causes oxidative stress and accelerates aging. Glutathione is conjugated with various activity-increasing substances and excreted in urine through the action of enzymes such as GST, thereby removing toxic substances from the body and improving sarcopenia. In addition, ROS is an important signaling molecule for the performance of normal cell functions, but abnormally increased ROS can cause cell death by causing dysfunction and damage to muscle cells.

Example 1: In order to examine the effect of the Schisandra chinensis solid-state fermentation extract on ROS and glutathione (GSH) production, muscle atrophy-inducing cells were treated with 50, 100, and 200 μg/ml of the Schisandra chinensis solid-state fermentation extract of Example 1. After measuring the relative production amounts of glutathione (a) and ROS (b) compared to the control group, the results are shown in Figure 12.

According to this, looking at the amount of glutathione (GSH) production, the GSH concentration was found to decrease by 40% in the atorvastatin treated group compared to the control group, and in the group treated with 100 μg/ml of Schisandra chinensis solid phase fermented extract of Example 1, Recovery was confirmed to be 67.60±2.32%, and 73.67±4.59% in the 200 ㎍/㎖ treatment group. In addition, the amount of ROS produced in the atorvastatin treated group showed a tendency to increase to 178.56 ± 5.16% compared to the control group, and in the group treated with 50 μg/ml of Schisandra chinensis solid phase fermentation extract of Example 1, it was 140.28. ±2.82%, 108.35±7.50% in the 100 ㎍/㎖ treatment group, and 105.67±6.26% in the 200 ㎍/㎖ treatment group, showing that the amount of ROS produced was significantly suppressed in a concentration-dependent manner of the extract.

Experimental Example 6: Cell death factor expression analysis

In general, free radicals are a key cause of sarcopenia in aging skeletal muscles, which reduces the function of mitochondria in muscle cells and eventually causes cell death. In order to determine the effect of the Schisandra chinensis solid-phase fermentation extract of Example 1 on apoptosis of muscle cells, the mRNA expression levels of bax, Bcl-2, caspase-3, and major regulators related to apoptosis were measured. RT-qPCR analysis was performed in the same manner as the method, and the results are shown in Figure 13. According to this, in the atorvastatin treated group, the mRNA expression levels of caspase-3 and Bax were increased, and the expression of Bcl-2 was decreased. In addition, the ratio of Bax/Bcl-2 showed that the amount of mRNA expression decreased in a concentration-dependent manner. Therefore, it can be seen that the solid-phase fermented extract of Schisandra chinensis in Example 1 is effective in preventing apoptosis of muscle cells.

In addition, in order to examine the expression level of proteins related to apoptosis of the solid-state fermented extract of Schisandra chinensis in Example 1, C2C12 myotube cells were treated with the solid-state fermented extract of Schisandra chinensis and atorvastatin, and then the apoptosis-related bax , bcl-2, caspase-3, and cleaved-caspase-3 proteins were measured through western blot. Specifically, C2C12 cells were treated with the sample at a concentration of 200 μg/ml and then treated with atorvastatin for 24 hours to induce muscle loss. Each cell was washed three times with 1×PBS and lysed with 0.1 ml of lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 μg/ml aprotinin). Proteins were separated by centrifugation at 12,000 rpm for 20 minutes, and the concentration of each separated protein was quantified in the protein analysis solution. Then, 30 μg protein was mixed with 5×sample buffer and separated through 8-15% SDS-PAGE. . The separated proteins on the gel were transferred to the NC membrane, and each membrane was blocked with 5% BSA for 1 hour at room temperature. Primary antibodies related to differentiation, apoptosis, and muscle loss were added to the membrane, reacted overnight at 4°C, and then washed three times with TBS containing 0.05% Tween. After adding anti-IgG conjugated HRP antibody to the membrane again, reacting at room temperature for 1 hour, washing three times with TBS (1×TBS) containing 0.05% Tween, and using ECL solution, ChemiDocTM touch imaging system (BioRad, California, USA) was used to analyze the results and the results are shown in Figure 14.

According to this, when comparing the expression levels of cleaved-caspase-3, caspase-3, and Bax/Bcl-2 in the atorvastatin treatment group with the control group, the expression levels of proteins related to apoptosis were significantly higher. increased, and in the group treated with Schisandra chinensis solid phase fermentation extract of Example 1, significant recovery was confirmed in the extract treatment group (***p<0.001 vs. CTL; ##p<0.01 and ###p< 0.001 vs. ATV treatment group).

[Experimental example: Extracellular experiment]

(1) Preparation and adaptation of experimental animals

The experimental animals used in the extracellular experiments were 6-week-old male C57BL/6 mice purchased from Duyeol Biotech (Seoul, Korea), and maintained at a temperature of 22±2°C, relative humidity of 60%, and a 12-hour light/dark cycle for 1 week. It was adapted to the liver environment and then used in experiments. During the adaptation period, water and feed were allowed to be consumed without restrictions. The entire experiment period was conducted for a total of 3 weeks, and after the adaptation period, the untreated group (CTL group), the dexamethasone treated group (Veh, Dex group), and the dexamethasone + Schisandra chinensis solid-state fermentation extract treated group of Example 3 (PS group 100, 300 ㎎/kg), dexamethasone + Schisandra chinensis extract treatment group (SC group 300 mg/kg), and 5 animals each were randomly assigned. After anesthesia on the day of the end of the experiment, blood was collected from the abdominal vein and stored, and the liver tissue of the experimental animals was extracted and stored at -0°C. This study was approved by the Institutional Animal Care and Use Committee (IACUC, 22-059) of the Korea Institute of Oriental Medicine and was conducted in accordance with the guidelines of the Korea Institute of Oriental Medicine IACUC.

(2) Induction of muscular atrophy animal model

After the experimental animal adaptation period, to create a muscle atrophy model, the Dex group, PS group, and SC group were intraperitoneally administered 5 mg/kg of dexamethasone every day between 10 and 11 a.m. for 14 days, and the CTL group was administered the same dose of physiological saline. was administered. The drug administration group was administered dexamethasone orally once a day at a dose of 100 and 300 mg/kg from one week before the administration until the end of the experiment, and the control group, the CTL group and the Dex group, were administered the same dose of physiological saline. Body weight was measured at 3-day intervals before and after dexamethasone administration. Each control and experimental group was euthanized at the end of the experiment, and the quadriceps and tibialis anterior (TA) muscles were removed.

A schematic diagram of the animal experiment design according to this is shown in Figure 15.

Experimental Example 7: Analysis of body weight change in dexamethasone-induced muscular atrophy animal model

In order to investigate the improvement effect of Schisandra chinensis solid-state fermentation extract (PS) on muscle atrophy, changes in body weight were confirmed in mice with Dex-induced muscle atrophy, and the results are shown in Figure 16. According to this, in the control group administered only saline solution, it was confirmed that body weight increased as age increased, and in the Veh group administered Dex, body weight significantly decreased as age increased compared to the CTL group. . Meanwhile, in the PS group, which was the drug administration group, there was no increase in body weight in terms of drug concentration, but when compared to the Vhe group in which muscle atrophy was induced with Dex, a significant increase in body weight was found. Looking at the difference in body weight, the normal control CTL group showed a weight gain of 3.8 ± 0.81 g, while the muscle atrophy-induced Veh group showed a significant decrease in weight gain of 1.7 ± 0.72 g, PS The weight gain was confirmed to be 2.3±0.37g in the 100 mg/kg group and 2.4±0.21g in the 300 mg/kg group, and in the Schisandra chinensis extract (SC 300 mg/kg) administration group, which was the positive control group, the weight gain was 2.1±0.46g. , a significant increase was confirmed in all drug groups compared to the Veh group. (p<0.01, ***p<0.001 vs. CTL; *p<0.05, #p<0.05, ##p<0.01 and ###p<0.001 vs. Dex group)

Experimental Example 8: Analysis of influence on exercise ability

Grip strength test and mouse treadmill test were performed to determine the effect of Sanghwang mushroom-Schias chinensis extract (PS) on the exercise capacity of a dexamethasone-induced muscular atrophy mouse model.

- How to measure grip strength (Grip strength test)

All experimental animals had their grip strength measured the day before the end of the experiment. Grip strength was measured in grams using the Bioseb grip strength test (BIO-GS3; BIOScience and Experimental Biology, Florida, USA). A stainless steel T-bar was used attached to the gauge. The experimental animal was made to grip the T-bar of the experimental tool with both arms, and the grip strength was measured by pulling the tail at a constant speed (2 cm/sec) until the grip was released. Five measurements were obtained for each animal and the average value was calculated.

- Mouse running test (Treadmill test) method

In the mouse running test, speed, duration, and distance were measured by a treadmill machine (Panlab, Barcelona, Spain) and controlled using software (SeDaCom v2.0.02, Panlab, Barcelona, Spain). In this test, the adaptive walking speed was set to run at 10 cm/sec for 3 minutes, and the speed was increased to 5 cm/sec every 5 minutes until the maximum speed of 75 cm/sec was reached. The adaptive walking speed and running speed were applied equally to all groups, and electrical stimulation (1.1 mA) was applied behind each rail of the treadmill to force them to run. The exercise ability of each individual was evaluated by comparing the time to exhaustion. The time when exhaustion occurred was defined as the time when the front leg was placed on the rail and the hind leg was placed on the electric device for 3 seconds.

The results of the grip test measurement according to the above method are shown in Figure 17. According to this, in the Grip test, which indicates grip strength, the CTL group showed a grip strength of 149.4 ± 22.8 g, while the Veh group, in which muscle atrophy was induced, showed a decrease in grip strength to 110 ± 16.5 g. In addition, it was confirmed that the reduced grip strength in the Veh group recovered as the drug concentration increased to 144.8 ± 25.4 g in the PS 100 administration group and 162 ± 16.0 g in the PS 300 administration group, and 153.8 ± 22.1 in the SC administration group, which was the control drug group. g, it was found that this also resulted in better recovery than the control group, Veh group.

Meanwhile, the results of the mouse running test (Treadmill test), which is an endurance test until fatigue during exercise, are shown in Figure 18. According to this, when induced to walk for an average of 5 minutes, the CTL group, which is a normal group, recorded 4.70 ± 0.49 min, while the Veh group, which is a muscle atrophy induction model, measured a relatively low 3.47 ± 0.94 min, and the PS group recorded 3.85 ± 3.85 min. It was recorded as 0.8 min, 4.29±0.48 min, and 3.85±0.84 min in the SC group, showing a significant difference from the Veh group. (p<0.01, ***p<0.001 vs. CTL; *p<0.05, #p<0.05, ##p<0.01 and ###p<0.001 vs. Dex group)

Experimental Example 9: Effect on quadriceps protection

Since the thickness and weight of the muscle are important indicators in the muscular atrophy animal model, a photograph of the femoral muscle removed after euthanasia is shown in Figure 19, and the weight and thickness were confirmed, and the results are shown in Figure 20.

According to this, the size of the thigh muscle was measured after autopsy, and it was found that the size of the thigh muscle decreased in the Vhe group, a muscle atrophy model induced by dexamethasone, compared to the CTL group, a normal control group, and the thickness and weight of the thigh muscle. When comparing the thickness, an increase in muscle thickness was seen in a drug concentration-dependent manner in the drug-administered PS group, but a concentration-dependent increase in weight could not be confirmed. However, when compared to the Veh group, the thickness and weight of the femoral muscles were found to increase in the PS group and the SC group, which is the drug comparison control group. ( **p<0.01, ***p<0.001 vs. CTL; * p<0.05, #p<0.05, ##p<0.01 and ###p<0.001 vs. Dex group)

Experimental Example 10: Analysis of morphological changes in muscle

A significant change in skeletal muscle atrophy in animal models of atrophy is characterized by a decrease in the size and number of muscle fibers (myofibers). In this study, in an animal model of muscle atrophy induced by dexamethasone, part of the tissues of the gastrocnemius and tibialis anterior (TA) muscles were fixed in 10% paraformaldehyde (Sigma Aldrich, St. Louis, MO, USA) and then paraffinized. Embedded in , the tissue was cut to a thickness of 3 ㎛ with a microtome (Thermo fisher scientific, Germany), stained with H&E, and histopathological changes were observed at 200x magnification using an optical microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan). did. The resulting optical microscope image is shown in Figure 21, where the upper part is a cross-sectional image of the muscle tissue, and the lower part is a longitudinal cross-sectional image of the muscle tissue.

According to this, compared to the normal muscle shape observed in the CTL group, the overall structure of muscle fibers in the Dex group was irregularly deformed, muscle fibers were also atrophied, the thickness of muscle fibers was reduced, and nuclei of various sizes and increased number of nuclei were observed. was observed. In contrast, in the PS group, the structure of muscle fibers showed a normal arrangement in a dose-dependent manner, and the thickness of muscle fibers was also confirmed to be increased.

Experimental Example 11: Effect on mRNA expression related to muscle breakdown/generation

After inducing an artificial muscular atrophy animal model with dexamethasone, to determine what effect PS extract had on muscle cell production (Myogenin), protein degradation factors (Atrogin-1, MuRF1), and energy metabolism regulatory pathways (AMPK, PGC1α). We isolated mRNA from muscle tissue and examined its expression.

Specifically, to confirm changes in muscle atrophy, RNA was extracted from the quadriceps muscle using the RNeasy ^® mini kit (Aiagen, Hilden, Gerbany). The extracted RNA was quantified using a Spectrophotometer (Nanodrop), and complementary DNA (cDNA) was synthesized from 1 μg RNA using the Maxima frist strand cDNA synthesis kit for RT-qPCR (Thermo scientific, Waltham, USA). Polymerase chain reaction (PCR) cycles were performed 40 times with 10 ㎕ of PCR bio syGreen blue Mix (PCR Biosystems, Pennsylvania, USA), 19 ㎕ of a mixture containing 2 ㎕ of primer, and 1 ㎕ of cDNA. Information on the primers used in the polymerase reaction is shown in Table 3 below.

Target Primer Sequences Myogenin Forward 5'-AGTACATTGAGCGCCTACAG-3' Reverse 5'-GACGTAAGGGAGTGCAGATT-3' MuRF1 Forward 5'-TGCCTACTTGCTCCTTGTGC-3' Reverse 5'-CACCAGCATGGAGATGCAGT-3' Atrogin-1 Forward 5'-CACCAAACCCACAGAAAACAG-3' Reverse 5'-GGGTCAGAGGAAGAGATAAAGTTG -3' PGC1α Forward 5'-ACAAACGGCTCACTTTGTCC-3' Reverse 5'-GTGCCGGATGGAGTTCTTC-3' AMPK Forward 5'-AGCCCTTCCTTCTCTTGCTC-3' Reverse 5'-AGGATGCCTGAAAGCTTGA-3' GAPDH Forward 5'-CAGCCTCGTCCCGTAGAC A-3' Reverse 5'-CGCTCCTGGAAGATGGTGAT-3'

The results of mRNA expression analysis related to muscle breakdown/generation according to the above method are shown in Figure 22. According to this, Myogenin, a transcription factor involved in the creation of muscle cells, tended to decrease by about 50% when compared to the CTL group, a normal control group, and the Veh group, a muscle atrophy induction group, and when treated with PS extract. , it was found that the expression level increased in a concentration-dependent manner. In addition, the expression level of Atrogin-1 and MuRF1, which are factors involved in muscle cell protein degradation, was confirmed to be increased in the Veh group, and when treated with PS extract, the mRNA expression level was confirmed to be decreased in a concentration-dependent manner at most concentrations. In this regard, it was determined that the PS extract would be effective in muscle atrophy, and as a result of examining the expression levels of pAMPK and PGC1α, it was confirmed that the expression levels were increased in the PS extract administered group. (**p<0.01, ***p<0.001 vs. CTL; *p<0.05, #p<0.05, ##p<0.01 and ###p<0.001 vs. Dex group)

Although the embodiments of the present invention have been described above, those skilled in the art can add, change, delete or add components without departing from the spirit of the present invention as set forth in the patent claims. The present invention may be modified and changed in various ways, and this will also be included within the scope of rights of the present invention.

Claims (17)

Contains Schisandra chinensis fermented by Phellinus linteus (Accession number: KCCM 60261) as an active ingredient,
A pharmaceutical composition for preventing or treating any muscle disease selected from muscular atrophy and sarcopenia. According to paragraph 1,
The fermented product of Schisandra chinensis is a solid-state fermented product of Schisandra chinensis, and the solid-state fermented product of Schisandra chinensis is a pharmaceutical composition for preventing or treating muscle disease, characterized in that the solid-state fermented product is solid-state fermented in a state where the moisture content of Schisandra chinensis powder is 45 to 60 wt%. According to paragraph 2,
A pharmaceutical composition for preventing or treating muscle disease, characterized in that the moisture of Schisandra chinensis powder during solid-state fermentation is deep ocean water with a hardness of 80 to 250 ppm. According to paragraph 2,
The pharmaceutical composition for preventing or treating muscle disease, characterized in that the solid-phase fermented product of Schisandra chinensis is an extract of the solid-state fermented product of Schisandra chinensis extracted with an extraction solvent. According to paragraph 4,
The extraction solvent is selected from water, lower alcohols having 1-4 carbon atoms, mixed solvents of the lower alcohols and water, acetone, ethyl acetate, chloroform, butyl acetate, 1,3-butylene glycol, hexane, and diethyl ether. A pharmaceutical composition for preventing or treating muscle disease, characterized in that it is one of the following. delete According to paragraph 1,
The pharmaceutical composition for preventing or treating muscle disease is used to increase the expression of one or more types selected from muscle cell production-related factors Myo-D, Myogenin, MEF2, Myf5, Myf6, and pAMPK. According to paragraph 1,
The pharmaceutical composition for preventing or treating muscle diseases is characterized in that it inhibits the expression of one or more types selected from Atrogin-1, MuRF-1, FoxO3α, and Myostatin, which are factors related to muscle cell breakdown. (a) drying and pulverizing Schisandra chinensis to prepare Schisandra chinensis powder;
(b) mixing the Schisandra chinensis powder with an aqueous solution of sugars and then sterilizing it to prepare a sterilized Schisandra chinensis pumpkin mixture; and
(c) inoculating the sterilized Schisandra chinensis mixture with Phellinus linteus , KCCM 60261 and performing solid-state fermentation to produce Schisandra chinensis solid-state fermentation; among muscular atrophy and sarcopenia, including; Method for producing a pharmaceutical composition for preventing or treating any selected muscle disease. According to clause 9,
In step (b), the mixing of the saccharide aqueous solution is a method of producing a pharmaceutical composition for preventing or treating muscle disease, characterized in that the moisture content of the sterilized Schisandra chinensis mixture is 45 to 60 wt%, According to clause 9,
In step (b), the aqueous saccharide solution is a method of producing a pharmaceutical composition for preventing or treating muscle disease, characterized in that it contains deep ocean water with a hardness of 80 to 250 ppm. According to clause 9,
After step (c),
(d) preparing a solid-state fermented Schisandra chinensis extract by mixing the solid-state fermented product with an extraction solvent, extracting and filtering the Schisandra chinensis bark solid-state fermented extract. A food composition for improving muscle strength containing as an active ingredient a fermented product of Schisandra chinensis fermented by Phellinus linteus (Accession number: KCCM 60261). According to clause 13,
The fermented product of Schisandra chinensis is a solid-state fermented product of Schisandra chinensis, and the solid-state fermented product of Schisandra chinensis is a food composition for improving muscle strength, characterized in that the solid-state fermented product is solid-state fermented in a state where the moisture content of the Schisandra chinensis powder is 45 to 60 wt%. (a) drying and pulverizing Schisandra chinensis to prepare Schisandra chinensis powder;
(b) mixing the Schisandra chinensis powder with an aqueous solution of sugars and then sterilizing it to prepare a sterilized Schisandra chinensis pumpkin mixture; and
(c) inoculating the sterilized Schisandra chinensis mixture with Phellinus linteus , KCCM 60261 and performing solid-state fermentation to produce a solid-state fermented Schisandra chinensis plant. A method for producing a food composition for improving muscle strength, comprising: A health functional food for improving muscle strength containing the food composition for improving muscle strength according to any one of claims 13 and 14. (a) drying and pulverizing Schisandra chinensis to prepare Schisandra chinensis powder;
(b) mixing the Schisandra chinensis powder with an aqueous solution of sugars and then sterilizing it to prepare a sterilized Schisandra chinensis pumpkin mixture; and
(c) inoculating the sterilized Schisandra chinensis mixture with Phellinus linteus , KCCM 60261 and performing solid-state fermentation to produce a solid-state fermented Schisandra chinensis plant; A method of manufacturing a health functional food for improving muscle strength, comprising: