KR102156540B1 - Composition for Preventing or Improving Muscle Atrophy Comprising Kukoamine A and Kukoamine B - Google Patents

Abstract

The present invention relates to a composition for preventing or improving skeletal muscle atrophy, wherein the composition for preventing or improving skeletal muscle atrophy containing gucoamine A and gucoamine B of the present invention as active ingredients It reduces, induces the synthesis of proteins involved in muscle fiber formation, and has high safety, and can be usefully used to prevent, improve and treat skeletal muscle atrophy.

Description

Composition for preventing or improving skeletal muscle atrophy containing Gukoamine A and Gukoamine B as active ingredients {Composition for Preventing or Improving Muscle Atrophy Comprising Kukoamine A and Kukoamine B}

The present invention relates to a composition for preventing or improving skeletal muscle atrophy, and more particularly, to a pharmaceutical composition and a food composition for preventing or improving skeletal muscle atrophy containing gucoamine A and gucoamine B as active ingredients.

In our body, the skeletal muscle consists of two main, white fast muscle fibers, active muscle (Type IIb) used to create movement, and red slow muscle fibers, which maintains a posture with weak strength for a long time. It can be divided into posture maintenance muscles (Type I). Among fast muscle fibers, Type IIa, which has the characteristics of slow muscle fibers, also exists.

When these muscle masses decrease due to various causes, the vicious cycle of musculoskeletal degeneration begins on the basis of weakening of muscle strength for physical activity. Decreased walking speed and weakened grip are the main symptoms and indicators of muscle mass loss, which may additionally lead to falls, fractures, joint damage, metabolic disorders, and cardiovascular disease. This decrease in muscle mass can be visually represented by a decrease in the volume of muscle fibers, that is, muscle atrophy.

Decreased muscle mass can occur naturally as the body ages, and appears as a cause of muscle unuse or lack of exercise, or other pathological conditions (cachexia, sepsis, starvation, chemotherapy, excessive exposure to stress hormones). ) May be the cause and may occur secondary. This can be combined with muscle cell atrophy due to anti-anabolic and catabolic action of Type I and II muscle fibers.

In many cases, stress hormones (Cortisol, Glucocorticoids, inner GCs) in our body cause molecular biological changes in muscle fibers and are directly or indirectly involved in anti-anaphylactic and catabolic processes. GCs play a role in inhibiting the PI3K/Akt/mTOR pathway as an anti-anabolic action, which inhibits the activities of downstream effectors 4E-BP1 and S6K1, and thus eIF4G (Eukaryotic translation initiation factor 4G) and It blocks the operation of eIF4E (Eukaryotic translation initiation factor 4E). This means that it inhibits the mRNA translation process for protein synthesis, and appears as muscle fiber atrophy due to inhibition of muscle fiber synthesis and protein degradation.

GCs not only inhibit muscle synthesis, but also play a role in inducing muscle atrophy by breaking down proteins. It expresses genes-Atrogenes (Atrogin-1, MuRF-1) that induce atrophy according to the mechanism leading to'PI3K/Akt -> FOXO activation and GSK3 inactivation', and these genes are represented by the Ubiquitin-Proteasome system. It induces protein degradation.

Untreated muscle loss can itself be a fatal health problem, and it can also cause harmful metabolic diseases and cause secondary problems that rapidly decrease activity. This can be a particularly serious problem in the elderly who already have age-related deficiencies in muscle mass, so there is a very high social demand to solve this problem.

Kukoamine A can be isolated from Lycium chinense or Potato peel, and its research is actively underway for various indications. The following prior art discloses the contents of various uses using gucoamine A. Prior Art 1 (Ponasik, J. et al., Biochemical Journal , 311(2), 371-375, 1995) relates to the inhibitory effect of gucoamine A and other hydrophobic acylpolyamines on Crithidia fasciculate trypanothione reductase. Technique 2 (Zhang, Y. et al., Neurochemical research , 41(10), 2549-2558, 2016) relates to the neuroprotective effect of gucoamine A on rat brain injury induced by oxidative stress and neuronal apoptosis. Prior Art 3 (Hu, XL et al., Biochimica et Biophysica Acta (BBA)-General Subjects , 1850(2), 287-298, 2015) is a method for oxidative stress related to N-methyl-D-aspartate receptors. It relates to the neuroprotective effect of gucoamine A.

Prior Art 4 (Hu, XL et al., Biochimica et Biophysica Acta (BBA)-General Subjects , 1850(2), 287-298, 2015) relates to the lipoxygenase inhibitory effect of gucoamine A derivatives. Technology 5 (Liu, J. et al., Neurochemistry international , 107, 191-197, 2017) relates to the ischemic cerebral neuroprotective effect of gucoamine A through antioxidant and anti-apoptotic, prior art 6 (Hu, X. et al., Neuropharmacology , 117, 352-363, 2017) relate to the neuroprotective effect of Kukoamine A in a neurotoxin-induced-Parkinson model through inhibition of apoptosis and induction of autophagy.

Prior Art 7 (Zhang, Y. et al., Neurotoxicity research , 31(2), 259-268, 2017) is based on the inhibitory effects of nF-kB and AP-1 in rats with radiation-induced neuritis. Min A confirmed the hippocampal nerve recovery effect, and prior art 8 (Li, G. et al., Biomedicine & Pharmacotherapy , 89, 536-543, 2017) shows the fatty liver of gucoamine A through downregulation of Srebp-1c. And insulin resistance inhibitory effect, prior art 9 (Wang, Q. et al., Scientific reports , 6, 2016) is a migration inhibitory effect and epithelial effect of gucoamine A through induction of malignant glioma cell growth and apoptosis. -Regarding the effect of reducing metastasis between germ layers, the prior arts do not disclose the effect and use of Gucoamine A on the prevention and improvement of muscle atrophy, and are not related to the present invention.

Accordingly, the present inventors have made diligent efforts to find a composition for preventing and treating skeletal muscle atrophy (Muscle Atrophy, Muscle Dystrophy), and as a result, Gukoamine A and Kukoamine B or their pharmaceutically acceptable It was confirmed that a pharmaceutical composition for preventing or improving skeletal muscle atrophy containing a salt as an active ingredient can prevent, improve, and treat skeletal muscle atrophy, and has high safety, and the present invention was completed.

An object of the present invention is to provide a composition capable of preventing and improving skeletal muscle atrophy.

Another object of the present invention is to provide a food composition for preventing or improving skeletal muscle atrophy containing as an active ingredient Gukoamine A and Kukoamine B or a pharmaceutically acceptable salt thereof.

In order to achieve the above object, the present invention is a skeletal muscle containing as an active ingredient at least one selected from the group consisting of Kukoamine A, Kukoamine B and its pharmaceutically acceptable salts. It provides a pharmaceutical composition for preventing or treating atrophy.

The present invention is also for preventing or improving skeletal muscle atrophy containing as an active ingredient at least one selected from the group consisting of Kukoamine A, Kukoamine B and its pharmaceutically acceptable salts Provide a food composition.

The present invention also provides a method for preventing or improving skeletal muscle atrophy comprising administering to an individual Gukoamine A and Kukoamine B or a pharmaceutically acceptable salt thereof.

The composition for preventing or improving skeletal muscle atrophy containing Gukoamine A and Gukoamine B of the present invention as active ingredients reduces the activity of proteins involved in muscle fiber atrophy and induces the synthesis of proteins involved in muscle fiber formation. It is highly safe and can be usefully used to prevent, improve, and treat skeletal muscle atrophy.

1 is a result of (A) HPLC chromatogram of the phalanges extract of the present invention and (B) gucoamine A and B of the present invention by HPLC-PDA-Mass.
2 is a result of HPLC chromatogram of gucoamine A and gucoamine B of the present invention.
3 is a result of observing the change in cell thickness according to the treatment concentration of Gukoamine A and Gukoamine B of the present invention.
4 is an image photographing C2C12 cells treated with gucoamine A according to the concentration of the present invention.
Figure 5 is a result of observing the expression of muscle atrophy factors (MuRF-1, Atrogin-1) according to the concentration of Gukoamine A treatment of the present invention.
6 is a result of observing the mRNA expression level of the muscle atrophy factor (Atrogin-1, MuRF-1) according to the concentration of Gukoamine A treatment of the present invention.
7 is a result of observing the expression of the signaling factor according to the treatment of Gukoamine A of the present invention.
8 is a result of confirming the mTOR activation effect of gucoamine A of the present invention.
9 is a result of confirming the inhibition of competition of rapamycin of gucoamine A of the present invention.
10 is a result of observing the change in cell thickness according to the concentration of Gukoamine B treatment of the present invention.
11 is a result of observing the expression of muscle atrophy factors (MuRF-1, Atrogin-1) according to the treatment of Gukoamine B of the present invention.
12 is a bar graph showing the results of observing the expression of muscle atrophy factors (MuRF-1, Atrogin-1) according to the treatment of Gukoamine B of the present invention.

In the present invention, it was confirmed that Kukoamine A induces the synthesis of proteins involved in the formation of muscle fibers through activation of the mTOR neurotransmitter pathway, and Kukoamine B increases the thickness of the root canal cells. It was confirmed to increase and decrease the activity of proteins involved in muscle fiber atrophy.

Accordingly, the present invention, in one aspect, Gukoamine A (Kukoamine A), Gukoamine B (Kukoamine B) and skeletal muscle atrophy containing one or more selected from the group consisting of a pharmaceutically acceptable salt thereof as an active ingredient It relates to a pharmaceutical composition for prophylaxis or treatment.

Gucoamine A of the present invention is represented by the following formula (1).

[Formula 1]

Gucoamine B of the present invention is represented by the following formula (2).

[Formula 2]

In the present invention, the term "muscular atrophy" refers to a decrease or contraction of muscle cells as the metabolic rotation of muscle synthesis and muscle breakdown is biased toward degradation, resulting in a decrease in muscle mass. Muscular atrophy is largely divided into those caused by a long-term stable condyle or fracture, etc., due to fixation of the cast, disease, and aging. Therefore, "muscular atrophy suppression" refers to suppressing the decrease in the function of the exercise machine or the decrease in muscle mass caused by the above cause.

In the present invention, muscle refers to skeletal muscle cells, and cardiac muscle is not included. Heart muscle is composed of mononuclear cells, and skeletal muscle is different in that there are multiple nuclei in one cell (Lee Jin-yi Kang and 21 others, Human Physiology 9th Edition, Life Science, p. 284, 2016). In addition, skeletal muscle cells atrophy occurs by the treatment of glucocorticoids such as dexamethasone, but the heart muscle has a difference in bringing about hypertrophy by treatment with these glucocorticoids. It can be seen that there is a clear difference (Kelly, FJ, & Goldspink, DF, Biochemical Journal , 208(1), 147, 1982).

In the present invention, "muscle degradation" means that the expression of genes related to the Akt pathway, cathepsin system, ubiquitin-proteasome system, autophagy system, etc. is induced, thereby promoting the degradation/catabolization of muscle fiber proteins. More specifically, it refers to the enhancement of muscle fiber protein degradation by inducing the expression of the Mstn gene or the ubiquitin-proteasome-related genes (Atrogin-1, MuRF1, Foxo1, etc.), which is one of the myoprotein degradation pathways.

In the present invention, "muscle synthesis" means that the synthesis/synthesis of muscle fiber proteins is promoted. More specifically, it means to induce activation of the kinase complex mTOR, which is a translation control factor in skeletal muscle, by suppressing the expression of the Mstn gene or the Redd1 gene, thereby promoting the synthesis of muscle proteins.

In the present invention, the composition may be characterized in that it inhibits the expression of any one or more in the group consisting of proteins involved in muscle atrophy, Atrogin-1, MuRF-1, Myostatin and SIRT1, and the composition It may be characterized by increasing the expression of any one or more in the group consisting of the proteins involved, PI3K, Adenosine A1R, Akt1 and TRPV4, but is not limited thereto.

Gukoamine A and Gukoamine B of the present invention inhibit proteins that can be muscle fiber atrophy factors, and increase the activity of proteins involved in muscle protein synthesis.

In the present invention, the expression includes not only the expression of the protein, but also the expression of the mRNA.

In the present invention, the Gukoamine A and Gukoamine B may be characterized in that they are extracted and separated from Lycium chinense or Potato peels, but are not limited thereto.

In the present invention, the phalanx blood used for the extraction of gucoamine A and gucoamine B is collected after passing the mouth and dried in the sun. It is recommended that the skin is large and thick, and the inner side is clean.

In the present invention, the Gukoamine A and Gukoamine B can be separated from Lycium chinense, which is the root bark/root bark (rhizodermis) of Goji chinense, but components of the phalanx cortex causing muscle atrophy prevention and improvement. Since A is also included in the root or branch of the wolfberry, its source is not limited to phalanx or potato peel.

In the present invention, the extract is extracted with a solvent selected from the group consisting of a mixture of water (H ₂ O), carbon atoms 1-4 lower alcohol and water (H ₂ O) with a carbon number of 1 to 4 lower alcohol The solvent may be characterized by being selected from the group consisting of water, methanol, ethanol, propanol, butanol, aqueous methanol, aqueous ethanol, aqueous propanol, and aqueous butanol, but is not limited thereto. .

The phalanges and potato peel extracts used to separate the gucoamine A and gucoamine B of the present invention include a lower alcohol having 1-4 carbon atoms, an aqueous alcohol solution, or water and phalanges or potato peels of 1:3 to 3: It may be one volume ratio, but is not limited thereto. Depending on the mixing ratio, the extraction efficiency of gucoamine A and gucoamine B components may vary.

In the present invention, the extraction may be characterized in that it is obtained by adding an acid for food and beverage purposes, and the acids are acetic acid, citric acid, malic acid. , Succinic acid, fumaric acid, tartaric acid, amino acid, and lactic acid may be selected from the group consisting of, but are not limited thereto.

In the extraction of phalanges or potato skins for the separation of gucoamine A and gucoamine B of the present invention, lower alcohol having 1-4 carbon atoms, aqueous alcohol solution or water in acetic acid, citric acid, malic acid (Malic acid), succinic acid, fumaric acid, tartaric acid, amino acid, lactic acid, and the like may be added. Using such an extraction method may increase the extraction efficiency of gucoamine A and gucoamine B. However, if such extraction is performed for a long time, the solvent instability of gucoamine A and gucoamine B may increase and the yield of the present component may be lowered. Therefore, it is necessary to adjust the extraction time appropriately.

In the present invention, the skeletal muscle atrophy is insoluble muscle atrophy (disuse atrophy of muscles), the absence of mechanical stimulation and denervation of skeletal muscle, cachexia, drug induced muscle atrophy, nutrient chamber It may be characterized in that at least one selected from the group consisting of Malnutrition induced muscle atrophy and muscular dystrophy, but is not necessarily limited thereto.

In the present invention, the term'disuse atrophy of muscles' includes reducing the thickness of the muscles due to activity restrictions due to aging, disease, etc., a long bed life, performing a splint, etc., and peripheral nerve damage, It is accompanied by various clinical diseases such as cancer and sepsis, as well as long-term bed stability, plaster fixation of limbs, and aging process. In general, muscular atrophy is accompanied by physiological, histochemical and biochemical changes due to a decrease in muscle protein, and can lead to intrinsic dysfunction of skeletal muscle.

In the present invention,'denervation-dominated muscle cells' result in a decrease in mitochondrial quantity and dysfunction, and increase in mitochondrial ROS production, resulting in an increase in apoptosis of muscle cells due to oxidative damage. Can be

In the present invention, "cachexia" is one of the symptoms of high systemic weakness that can be seen at the end of cancer, tuberculosis, and hemophilia, and rapid contraction may occur.

In the present invention, "muscular dystrophy" is a disease in which muscle atrophy and weakening of muscle strength are caused by a member of the dystrophin-glycoprotein complex, which is a component of the rhabdomyoplasmic membrane, due to genetic mutation.

In the present invention, the muscle atrophy includes muscle atrophy caused by cortisol, a substance that can be secreted from the body due to various acute stresses, and steroids caused by adverse reactions of synthetic adrenal corticosteroids Sexual myopathy, muscle parenchyma damage, etc.

In the present invention, the composition may be characterized in that it has an effect of preventing and improving the reduction of muscle fiber bundles, inflammatory cell infiltration or local fibrosis of the muscle, and the composition activates intracellular protein synthesis mechanisms including mTOR. It may be characterized by letting, but is not limited thereto.

In one embodiment of the present invention, as a result of treatment with dexamethasone and gucoamine A on myotube cells C2C12, gucoamine A shows remarkably excellent muscle atrophy recovery rate for dexamethasone, a muscle atrophy material, and is concentration-dependent. , It was confirmed that dexamethasone antagonizes the increased activity of muscle atrophy factors. In addition, it was confirmed that protein synthesis involved in muscle fiber formation was induced through activation of the mTOR neurotransmitter pathway.

In addition, as a result of treatment with dexamethasone and gucoamine B on the root canal cells C2C12, it was confirmed that gucoamine B showed remarkably excellent muscle atrophy recovery rate, was concentration-dependent, and reduced the activity of muscle atrophy factors.

In the present invention, dexamethasone is one of the biosynthetic substances of glucocorticoids, and increases the expression of Atrogin-1 and MuRF-1, which are involved in muscle breakdown in muscle tissue, and inhibits PI3K and Akt activities, thereby inhibiting FoxO and E3 ligase. Promotes muscular atrophy by increasing activity.

In the present invention, ursolic acid (UA) activates insulin and insulin-like growth factor-1 (IGF-1), which play a major role in muscle formation, and muscle atrophy in which muscles are weakened by disease or aging. It is a substance that inhibits genetic changes that cause atrophy).

In the present invention, the "Akt pathway" is a generic term for a signaling pathway mediated by Akt, a kinase involved in the control of various cellular functions. Akt activity is regulated due to various stimuli such as nutrition, growth factors, and exercise. Activated Akt activates ribosomal protein S6 kinase (S6K) involved in muscle synthesis through mTOR (mammalian target of rapamycin). In addition, activated Akt is known to indirectly inhibit muscle degradation by inhibiting Foxo1, which promotes muscle degradation.

In the present invention,'Atrogin-1' and'MuRF1 (muscle RING-finger protein-1)' are ubiquitin ligase involved in the ubiquitin-proteasome system, which is one of the muscle degradation pathways, and are expressed in skeletal muscle. Is known.

In the present invention, "Foxo1" refers to a forkhead box protein O1. Foxo1 is usually localized in the cytoplasm in a phosphorylated state, but is transferred into the nucleus by dephosphorylation and functions as a transcription factor. Increasing the expression of Foxo1 is commonly recognized in muscle atrophy, and it is known to be involved in various mechanisms such as protein degradation by the ubiquitin-proteasome system, enhancement of autophagy, and further inhibition of protein synthesis.

The composition for preventing or improving skeletal muscle atrophy of the present invention is used for prevention or treatment of decreased motor function or motor disorder. For example, it includes, but is not limited to, fixation of casts due to long-term stable condyles or fractures, and prevention or treatment of motor disorders or motor syndromes caused by diseases, aging, and the like. This use may be a human or non-human animal, and may be a therapeutic use or a non-therapeutic use. Here, “non-therapeutic” refers to a concept that does not include medical actions, that is, treatment actions on the human body following treatment.

The composition for preventing or improving skeletal muscle atrophy of the present invention is used for the prevention or treatment of drug-induced muscle atrophy. For example, it includes, but is not limited to, the prevention or treatment of muscle atrophy caused by long-term intake of steroid hormones. This use may be a human or non-human animal, and may be a therapeutic use or a non-therapeutic use.

The pharmaceutical composition of the present invention may further include an appropriate carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions. Compositions containing a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration may include tablet pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, or lactose in one or more compounds. (lactose), gelatin, etc. can be mixed to prepare. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have. Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

In addition, the pharmaceutical composition of the present invention is not limited thereto, but tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations And it may have any one formulation selected from the group consisting of suppositories.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenous, subcutaneous, intraperitoneal or topical application) according to a desired method, and the dosage is the condition of the patient (or individual) and It depends on the body weight, the degree of disease, the form of the drug, the route of administration and the time, but may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, drug activity of the patient, Sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or administered in combination with another therapeutic agent, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient in the body, inactivation rate and excretion rate, the type of disease, and the drug to be used in combination. 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight may be administered daily or every other day, or divided into 1 to 3 times a day. However, since it may increase or decrease according to the route of administration, the severity of obesity, sex, weight, age, etc., the dosage amount does not limit the scope of the present invention in any way.

The present invention may be a pet food or animal feed processed as pet food, and an animal medicine.

The composition of the present invention may be used alone for the prevention and treatment of skeletal muscle atrophy, or in combination with surgery, hormone therapy, chemotherapy, and methods using biological response modifiers.

In another aspect, the present invention prevents skeletal muscle atrophy containing as an active ingredient at least one selected from the group consisting of Kukoamine A, Kukoamine B, and a pharmaceutically acceptable salt thereof. It relates to an improvement food composition.

In the food composition for preventing or improving skeletal muscle atrophy according to an embodiment of the present invention, the food composition may be used as a health functional food that can be consumed on a daily basis to inhibit muscle aging or improve exercise function.

In the present invention, the term'health functional food' refers to Kukoamine A and Kukoamine B of the present invention or a pharmaceutically acceptable salt thereof, such as beverages, teas, spices, gums, confectionery, etc. It is a food that is added to the food ingredients of, or is prepared as encapsulated, powdered, or suspension. When ingested, it means that it has a specific effect on health, but unlike general drugs, when taking a drug for a long time using food as a raw material. It has the advantage of no side effects that may occur. 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 food composition according to the present invention includes all foods such as beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, ice creams, alcoholic beverages, and vitamin complexes. Depending on the type of food, it cannot be uniformly defined, but it may be added within a range that does not impair the original taste of the food, and is usually 0.01 to 50% by weight, preferably 0.1 to 20% by weight, based on the target food. In addition, in the case of food in the form of granules, tablets, or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 5 to 100% by weight.

In the present invention, it may be characterized in that the food composition further comprises a food additive acceptable food additives.

The functional food of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used.

The ratio of the natural carbohydrate is preferably selected from the range of 0.01 to 0.04 parts by weight, preferably about 0.02 to 0.03 parts by weight per 100 parts by weight of the health food of the present invention.

In addition to the above, the functional food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol, carbonates used in carbonated beverages, and the like. In addition, the functional food of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. Although the ratio of these additives is not very important, it is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention.

In the present invention, it may be characterized in that it further comprises an extract derived from Lycium chinense, an extract derived from Potato peels, and/or an extract fraction in the food composition.

In another aspect, the present invention comprises administering to an individual at least one selected from the group consisting of Kukoamine A, Kukoamine B, and a pharmaceutically acceptable salt thereof. A method of preventing or improving skeletal muscle atrophy can be provided.

The term "individual" used in the present invention may mean all animals including humans who have or are likely to develop skeletal muscle atrophy disease. The animals may be mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, etc. in need of treatment for symptoms similar to humans, but are not limited thereto.

Specifically, the method of preventing or improving the present invention may include administering the composition in a pharmaceutically effective amount to an individual who has or is at risk of developing skeletal muscle atrophy.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention is oral or parenteral as long as it can reach the target tissue. It can be administered through a variety of routes.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1: Extraction and purification of Gucoamine A and Gucoamine B

Lycium Radicis Cortex (地骨皮, Lycium Radicis Cortex) used in this experiment was purchased from Deokhyeondang (Gyeongdong Market, Seoul), and the specimen (HYUP-LCR-001) was stored at Hanyang University College of Pharmacy. 3 Kg of the crude phalanges powder was extracted with a solvent having an ethanol:water (50:50, v/v) composition to obtain 156 g of a crude extract. The crude phalanges extract was subjected to gradient elution with water and ethanol, and fractionated using Diaion HP-20 column chromatography.

The active fraction was subjected to Sephadex LH-20 open column chromatography, and HPLC chromatograms were performed on 8 samples obtained. The mobile phase solvent was EtOH: DDW (10: 90, v/v) in a ratio, and the fractions were separated and collected using thin layer chromatography (TLC). This was again separated from gucoamine A and gucoamine B using Prep-HPLC (column: HECTOR (C18, 5micron, 250×21.2 mm, RS tech corporation, Korea).

Example 2: Cell line Mouse myotubes C2C12, preparation step for material treatment

The experimental cell line was obtained from the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center and induced division for a total of 2 days x 3 times for a total of 6 days. After sufficient division is completed, C2C12 is seeded in a 12-well cell plate and divided for 2 days. The medium used for the division was HyClone™ DMEM/HIGH GLUCOSE as a culture medium, and 10% fetal bovine serum (FBS) and 1% penicillin were added.

After the 12 Well Cell plate is sufficiently tightly filled, the medium for differentiation is treated for 6 days. Differentiation medium is replaced with new medium every 2 days. The medium required for differentiation is made of HyClone™ DMEM/HIGH GLUCOSE as a culture medium and 2% horse serum (HS) and 1% penicillin added.

The differentiated C2C12 myotubes were treated with a medium in which 1% Penicillin was added to HyClone™ for 24 hours.

Until this process, the preparation stage for the treatment of drug candidates on cells.

Example 3: Confirmation of the thickness of differentiated myotube cells C2C12 according to the treatment of muscle atrophy substances Dexamethasone and Kukoamine A

In order to confirm the thickness of the root canal cells C2C12 according to Gukoamine A treatment, the root canal cells C2C12 prepared in Example 2 were classified into three groups as follows and treated.

(1) Control-DMEM/HIGH GLUCOSE medium containing only 1% Penicillin was added to C2C12 after differentiation was completed for 48 hours.

(2) Negative Control-DMEM/HIGH GLUCOSE medium added with 1% Penicillin was prepared to have a composition of 1uM dexamethasone, and then treated in C2C12 where differentiation was completed for 48 hours.

(3) Negative control group + Gucoamine A treatment-Gucoamine A was treated for 48 hours in the same conditions as the negative control in (2) in a composition of 25 μM, 50 μM and 100 μM, respectively

Afterwards, to check the thickness of differentiated C2C12 myotube cells, each control group and experimental group treated for 48 hours were photographed using JuLi™ Stage, an automated cell imaging system, and photographed using Image J, a cell processing program. The thickness of each cell was measured, 50 cells per cell group were measured, and the average and standard error were calculated and the data were organized.

As a result, it was found that gucoamine A showed remarkably excellent muscle-atrophy recovery rate for dexamethasone, which is a muscle atrophy substance (FIG. 3).

In addition, as the concentration of gucoamine A increased, the thickness of the root canal cells also increased. Therefore, it can be seen that the increase in the thickness of the root canal cells is concentration-dependent for gucoamine A (Fig. 3).

The following description shows the results of comparing the thickness of the root canal cells as a result of treatment under four conditions.

(1) Compared to the basic nutrient medium administration group, Control, in the'nutrition medium + dexamethasone (Dex) 1μM' group, the thickness decreased by 23.4%, which is statistically very significant (p<0.005).

(2-1) In the group administered'nutrition medium + dexamethasone 1μM + gucoamine A 25μM' compared to the negative control group administered with'nutrition medium + dexamethasone 1μM', the thickness increased by 15.7%, which was statistically very significant (p<0.005 )Do.

(2-2) As a result of calculating the above value as a recovery rate (“Dex+Kukoamine A 25 μM”-Dex compared to Control-Dex), it is 0.517, that is, the damage recovery rate of Gukoamine A 25 μM can be expressed as 51.7%.

(3-1) In the group administered'nutrition medium + dexamethasone 1μM + gucoamine A 50μM' compared to the negative control group administered with'nutrition medium + dexamethasone 1μM', the thickness increased by 24.2%, which was statistically very significant (p<0.005 )Do.

(3-2) As a result of calculating the above value as a recovery rate (“Dex+Kukoamine A 50 μM”-Dex compared to Control-Dex), it is 0.790, that is, the damage recovery rate of Gukoamine A 50 μM can be expressed as 79.0%.

(4-1) In the group administered'nutrition medium + dexamethasone 1μM + gucoamine A 100μM' compared to the negative control group administered with'nutrition medium + dexamethasone 1μM', the thickness increased by 28.9%, which was statistically very significant (p<0.005 )Do

(4-2) As a result of calculating the above value as a recovery rate ('Dex+Kukoamine A 100 μM'-Dex compared to Control-Dex), it is 0.948, that is, the damage recovery rate of Gukoamine A 100 μM can be expressed as 94.8%.

Therefore, it can be seen that gucoamine A shows a remarkably excellent recovery rate of muscle atrophy with respect to dexamethasone and is concentration-dependent.

Example 4: Confirmation of the activity of muscle atrophy factor proteins (MuRF-1 and Atrogin-1) according to the treatment of the muscle atrophy substances Dexamethasone and Kukoamine A

Protein activity was measured for each control group and experimental group treated under the three conditions in Example 3 by Western blotting.

In addition, the biochemical activity of Gucoamine A was verified by measuring MuRF-1 and Atrogin-1, which are the final downstream effectors of the muscle atrophy pathway.

As a result, as shown in FIG. 5, when comparing the left and right data of Western blotting, muRF-1, which is a muscle atrophy factor, in the group treated with dexamethasone (right) compared to the group not treated with dexamethasone (left), and It was confirmed that the expression of Atrogin-1 was increased.

Comparing the right western blotting data, it was confirmed that the expression of muRF-1 and Atrogin-1, which are muscle atrophy factors, decreased as the concentration of gucoamine A increased.

Therefore, it can be seen that gucoamine A antagonizes the increase in the activity of the muscle atrophy factor caused by dexamethasone.

It was confirmed that when the dexamethasone-treated group was treated with 100 μM of gucoamine A, the mRNA expression levels of muRF-1 and Atrogin-1 were decreased. This can be said that the transcription factors of the corresponding genes were reduced by gucoamine A (FIG. 6).

In addition, in order to confirm the mTOR activation effect of Gucoamine A, it was observed using FACS. As a result, it can be seen that gucoamine A activates the process of synthesizing muscle fiber protein through mTOR, and in particular, it was confirmed that mTOR whose expression rate is improved by gucoamine A is a protein that is inhibited by Rapamycin. (Fig. 7).

Checking the data obtained with a flow cytometer, a high level of decrease in mTOR activity appeared in the group treated with dexamethasone relative to the control group, and that when Gucoamine A was treated as a drug, the value was corrected over a certain amount. Confirmed (Fig. 8).

In addition, in the group treated with dexamethasone, gucoamine A, and rapamycin, it was confirmed that the protein activity of gucoamine A was significantly reduced by rapamycin (FIG. 9). Therefore, it can be seen that the inhibitory effect on muscle atrophy by gucoamine A is through the protein synthesis pathway inhibited by rapamycin.

Through this, gucoamine A decreases dexamethasone-induced muscle atrophy or muscle dystrophy in a concentration dependent manner in the C2C12 in vitro assay, and that it passes through a protein synthesis pathway inhibited by rapamycin. Able to know.

Example 5: Confirmation of the thickness of differentiated myotube cells C2C12 according to the treatment of muscle atrophy substances Dexamethasone and Kukoamine B

In the same manner as in the experimental method in which Gukoamine A was treated in Example 3, the thickness of the root canal cells C2C12 according to Gukoamine B treatment was confirmed.

As a result, it was found that gucoamine B showed a very high muscle-atrophy recovery rate for dexamethasone. In addition, as the concentration of gucoamine B increases, the thickness also increases, and it can be seen that the concentration is dependent (FIG. 10).

In addition, it was confirmed that gucoamine B exhibits higher muscle-atrophy prevention and improvement activity compared to ursolic acid (UA), which is known as the existing positive control (FIG. 10).

The following description shows the results of comparing the thickness of the root canal cells as a result of treatment under four conditions.

(1) The thickness decreased by 23.4% in the'nutrition medium + dexamethasone 1μM' group compared to the basic nutrient medium administration group, Control, which is statistically very significant (p<0.005).

(2-1) In the group administered'nutrition medium + dexamethasone 1μM + gucoamine B 25μM' compared to the negative control group administered with'nutrition medium + dexamethasone 1μM', the thickness increased by 15.6%, which was statistically very significant (p<0.005 )Do.

(2-2) As a result of calculating the above value as a recovery rate (“Dex+Kukoamine B 25 μM”-Dex compared to Control-Dex), it is 0.512, which can be expressed as 51.2% of the damage recovery rate of Gukoamine B 25 μM.

(3-1) In the group administered'nutrition medium + dexamethasone 1μM + gucoamine B 50μM' compared to the negative control group administered with'nutrition medium + dexamethasone 1μM', the thickness increased by 19.0%, which was statistically very significant (p<0.005 )Do.

(3-2) As a result of calculating the above value as a recovery rate (“Dex+Kukoamine B 50 μM”-Dex compared to Control-Dex), it is 0.623, and the damage recovery rate of 50 μM Gukoamine B can be expressed as 62.3%.

(4-1) In the group administered'nutrition medium + dexamethasone 1μM + gucoamine B 100μM' compared to the negative control group administered with'nutrition medium + dexamethasone 1μM', the thickness increased by 21.9%, which was statistically very significant (p<0.005 )Do.

(4-2) As a result of calculating the above value as a recovery rate (“Dex+Kukoamine B 100 μM”-Dex compared to Control-Dex), it is 0.717, and the damage recovery rate of Gukoamine B 100 μM can be expressed as 71.7%.

Therefore, it can be seen that gucoamine B shows a remarkably excellent recovery rate of muscle atrophy with respect to dexamethasone and is concentration dependent.

Example 6: Confirmation of the activity of muscle atrophy factor proteins (MuRF-1 and Atrogin-1) according to the treatment of the muscle atrophy substances Dexamethasone and Kukoamine B

The activities of MuRF-1 and Atrogin-1 according to Gucoamine B treatment were confirmed in the same manner as in the experimental method in which Gucoamine A was treated in Example 4.

As a result, it was confirmed that when dexamethasone and gucoamine B were administered together compared to the group administered with dexamethasone alone, the activity of both muRF-1 and Atrogin-1, which are muscle atrophy factors, decreased (FIGS. 11 and 12).

Accordingly, it can be seen that gucoamine A and gucoamine B of the present invention exhibit remarkable effects in preventing and improving muscular atrophy.

Preparation Example 1: Preparation of a food composition for preventing or improving skeletal muscle atrophy

<Production Example 1> Preparation of pharmaceutical formulation

<1-1> Preparation of powder

Kukoamine A or 2 g of its pharmaceutically acceptable salt

1 g lactose

The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

<1-2> Preparation of tablets

Kukoamine A and Kukoamine B, 100 mg each

Corn starch 100 mg

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tablets were prepared by tableting according to a conventional tablet preparation method.

<1-3> Preparation of capsules

Kukoamine A or Kukoamine B 100 mg

Corn starch 100 mg

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, the gelatin capsules were filled according to a conventional capsule preparation method to prepare a capsule formulation.

<1-4> Preparation of ring

Kukoamine A and Kukoamine B 100 mg each

1.5 g lactose

1 g glycerin

0.5 g xylitol

After mixing the above ingredients, it was prepared so as to be 4 g per ring according to a conventional method.

<1-5> Preparation of granules

Kukoamine A and Kukoamine B or 150 mg of a pharmaceutically acceptable salt thereof

Soybean extract 50 mg

200 mg of glucose

Starch 600 mg

After mixing the above ingredients, 100 mg of 30% ethanol was added, dried at 60°C to form granules, and then filled into a cloth.

<Production Example 2> Preparation of food

<2-1> Preparation of flour food

For 100 parts by weight of flour, 10 parts by weight of each of Kukoamine A and Kukoamine B of the present invention are added to the flour, and bread, cakes, cookies, crackers and noodles are prepared using this mixture. Was prepared.

<2-2> Preparation of soup or gravies

For health promotion by adding 0.1 to 5.0 parts by weight of Kukoamine A and Kukoamine B or pharmaceutically acceptable salts thereof of the present invention to 100 parts by weight of soup or broth Meat products, noodle soups, or juices were prepared.

<2-3> Preparation of ground beef

Ground beef for health promotion was prepared by adding 10 parts by weight of Gukoamine A and Kukoamine B or a pharmaceutically acceptable salt thereof of the present invention to 100 parts by weight of ground beef I did.

<2-4> Manufacture of dairy products

For 100 parts by weight of milk ground beef, 5 to 10 parts by weight of Gukoamine A and Gukoamine B or a pharmaceutically acceptable salt thereof of the present invention are added to the milk, and , Various dairy products such as butter and ice cream were prepared by using the milk.

<2-5> Manufacturing of Seonsik

Brown rice, barley, glutinous rice, and adlay were gelatinized by a known method, dried, and then roasted, and then prepared into a powder having a particle size of 60 mesh with a grinder.

Black soybeans, black sesame seeds, and perilla seeds were also steamed and dried by a known method, and then roasted, and then prepared into a powder having a particle size of 60 mesh with a grinder.

Kukoamine A and Kukoamine B of the present invention are concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by spraying and drying with a hot air dryer is pulverized with a grinder to a particle size of 60 mesh to obtain dry powder. Got it.

The grains, seeds, and Gukoamine A and Kukoamine B, or a pharmaceutically acceptable salt thereof, were mixed in the following ratio to prepare them.

Cereals (35% by weight of brown rice, 16% by weight of adlay, 20% by weight of barley),

Seeds (perilla 7% by weight, black beans 8% by weight, black sesame 7% by weight),

Kukoamine A and Kukoamine B (3% by weight each),

Ganoderma lucidum (0.5% by weight),

Rehmannia (0.5 wt%)

<Production Example 3> Preparation of beverage

<3-1> Manufacturing of health drinks

Subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), water (75%), and Gukoamine A and Gukoamine B of the present invention (Kukoamine B) or 5 g of a pharmaceutically acceptable salt thereof was homogeneously mixed and sterilized at an instant, and then packaged in a small container such as a glass bottle or a plastic bottle to prepare it.

<3-2> Preparation of vegetable juice

Vegetable juice was prepared by adding 5 g of Kukoamine A and Kukoamine B of the present invention or a pharmaceutically acceptable salt thereof to 1,000 ml of tomato or carrot juice.

<3-3> Preparation of fruit juice

Fruit juice was prepared by adding 1 g of Kukoamine A and Kukoamine B of the present invention or a pharmaceutically acceptable salt thereof to 1,000 ml of apple or grape juice.

As described above, specific parts of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (14)

Gukoamine A (Kukoamine A), Gukoamine B (Kukoamine B), and a pharmaceutical composition for the prevention or treatment of skeletal muscle atrophy containing as an active ingredient at least one selected from the group consisting of a pharmaceutically acceptable salt thereof.
The pharmaceutical composition of claim 1, wherein the composition inhibits the expression of any one or more in the group consisting of Atrogin-1, MuRF-1, Myostatin, and SIRT1, which are proteins involved in muscle atrophy.
The pharmaceutical composition according to claim 1, wherein the composition increases the expression of any one or more in the group consisting of PI3K, Adenosine A1R, Akt1 and TRPV4, which are proteins involved in muscle synthesis.
The pharmaceutical composition according to claim 1, wherein the Gukoamine A and Gukoamine B are extracted and separated from Lycium chinense or Potato peels.
The method of claim 4, wherein the extract is extracted with a solvent selected from the group consisting of a mixture of water (H ₂ O), carbon atoms 1-4 lower alcohol and water (H ₂ O) with a carbon number of 1 to 4 lower alcohol Pharmaceutical composition, characterized in that obtained by obtaining.
The pharmaceutical composition according to claim 5, wherein the solvent is selected from the group consisting of water, methanol, ethanol, propanol, butanol, aqueous methanol, aqueous ethanol, aqueous propanol, and aqueous butanol.
The pharmaceutical composition of claim 5, wherein the extraction is obtained by additionally adding an acid for food and beverage purposes.
The method of claim 7, wherein the acid is acetic acid, citric acid, malic acid, succinic acid, fumaric acid, tartaric acid, amino acid, Pharmaceutical composition, characterized in that selected from the group consisting of lactic acid (Lactic acid).
The method of claim 1, wherein the skeletal muscle atrophy is disuse atrophy of muscles, the absence of mechanical stimulation and denervation of skeletal muscle, cachexia, drug induced muscle atrophy, nutrition. A pharmaceutical composition, characterized in that it is at least one selected from the group consisting of Malnutrition induced muscle atrophy and muscular dystrophy.
The pharmaceutical composition according to claim 1, wherein the composition has an effect of reducing muscle fiber bundles, preventing and improving inflammatory cell infiltration or local fibrosis of muscles.
The pharmaceutical composition of claim 1, wherein the composition activates an intracellular protein synthesis mechanism including mTOR.
Gukoamine A (Kukoamine A), Gukoamine B (Kukoamine B), and a food composition for preventing or improving skeletal muscle atrophy containing as an active ingredient at least one selected from the group consisting of a pharmaceutically acceptable salt thereof.
The food composition according to claim 12, further comprising a food additive acceptable for food.
The food composition of claim 12, further comprising an extract derived from Lycium chinense, an extract derived from Potato peels, and/or an extract fraction.

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