KR20220167012A - A composition for preventing or treating of sarcopenia containing novel compounds isolated from allium tuberosum - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating sarcopenia, and more specifically, to a composition for preventing, alleviating or treating sarcopenia containing a novel compound isolated from Allium tuberosum or a salt thereof. The compound acts as a regulator which regulates the PI3K/Akt/mTOR or Smad pathway and significantly induces the growth and differentiation of muscle cells, and thus is expected to be widely used as medicine, foods, and quasi-drugs for the prevention and treatment of sarcopenia.

Description

Composition for preventing or treating sarcopenia containing novel compounds derived from leek {A composition for preventing or treating of sarcopenia containing novel compounds isolated from allium tuberosum}

The present invention relates to a composition for preventing, improving or treating sarcopenia, and more particularly, to a composition for preventing, improving or treating sarcopenia comprising a novel compound isolated from leek or a salt thereof.

Aging causes a loss of skeletal muscle mass and strength, which is called sarcopenia. Sarcopenia is a lifelong process that begins at about the age of 30, and the amount of muscle tissue and the number and size of gold fibers gradually decrease, resulting in loss of muscle mass and strength. As a result, loss of mobility, falls, osteoporosis, and fractures result in decreased quality of life and decreased physical activity. Recently, the prevalence is increasing due to the increase in the elderly population, and as the burden of medical expenses increases due to this, the need for research that can lower it is emphasized. Therefore, many researchers are actively conducting research to find ingredients from natural products that can help increase and maintain skeletal muscle mass.

On the other hand, leek (Allium tuberosum) is a perennial herb belonging to the Liliaceae family, and is mainly cultivated and consumed in East and Southeast Asia, and has been used for the treatment of abdominal pain, diarrhea, hematemesis, and asthma in China. Studies on the pharmacological activity of chives include antidiabetic and hepatoprotective effects, lipid improvement effects by reducing serum cholesterol, triglyceride, low-density lipoprotein cholesterol and arteriosclerosis index, and cancer cell proliferation inhibition effects through apoptosis (KOREAN J.FOOD SCI.

As a result of intensive research on natural products for the treatment of sarcopenia, the present inventors isolated a novel compound from leek, and the compound acts as a regulator that regulates the PI3K/Akt/mTOR or Smad pathway, resulting in the growth and differentiation of muscle cells. It was confirmed that it can be usefully used as a composition for preventing and treating sarcopenia by significantly inducing.

Accordingly, an object of the present invention is to provide a novel compound represented by Formula 1 below.

[Formula 1]

In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating sarcopenia, comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, another object of the present invention is to provide a health functional food composition for preventing or improving sarcopenia, comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a novel compound represented by Formula 1 below.

[Formula 1]

In one embodiment of the present invention, the compound may have muscle loss inhibitory activity.

In another embodiment of the present invention, the compound can induce the growth and differentiation of muscle cells.

In another embodiment of the present invention, the compound can induce the growth and differentiation of muscle cells by activating the PI3K/Akt/mTOR signaling pathway or down-regulating the Smad signaling pathway.

In another embodiment of the present invention, the compound may be extracted and isolated from leek.

In addition, the present invention provides a pharmaceutical composition and a health functional food composition for preventing, improving or treating sarcopenia, comprising the compound or a pharmaceutically or food-acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the compound can induce the growth and differentiation of muscle cells.

In another embodiment of the present invention, the compound can induce growth and differentiation of muscle cells by activating the PI3K/Akt/mTOR signaling pathway or down-regulating the Smad signaling pathway.

It is expected that the compound can be widely used as pharmaceuticals, foods, and quasi-drugs for the prevention and treatment of sarcopenia by significantly inducing the growth and differentiation of muscle cells by acting as a regulator that regulates the PI3K/Akt/mTOR or Smad pathway do.

Figure 1 is a schematic diagram showing the process of separating the novel compound according to the present invention from the leek extract.
Figure 2 confirms the skeletal muscle cell differentiation activity of the novel compound according to the present invention, Figure 2a shows the result of confirming the cytotoxicity of the novel compound of the present invention, Figure 2b shows the result of confirming myoblast cell differentiation.
Figure 3 confirms the activation of the PI3K / Akt / mTOR signaling pathway related to skeletal muscle growth by the novel compound according to the present invention, Figure 3a confirms the change in the expression level of six proteins related to skeletal muscle differentiation using Western blot The results are shown, and FIGS. 3b to 3g show the results of quantifying the degree of expression level of each protein disclosed in FIG. 3a.
Figure 4 is a result of confirming that the novel compound according to the present invention down-regulates the Smad signaling pathway related to muscle growth and differentiation. Figure 4a confirms the expression levels of p-Smad2/3 and Smad4 proteins using Western blot. , Figures 4b to 4d show the results of quantifying each protein expression level disclosed in Figure 4a.
Figure 5 is a schematic diagram showing the skeletal muscle differentiation control pathway according to the mechanism of Myostatin and IGF-1.

The present inventors isolated a novel compound from leek, and the compound acts as a regulator that regulates the PI3K/Akt/mTOR or Smad signaling pathway to significantly induce the growth and differentiation of muscle cells, thereby preventing and treating sarcopenia. It was confirmed that it can be usefully used as a composition for use, and the present invention was completed.

Accordingly, the present invention provides a novel compound. The novel compound according to the present invention is composed of the chemical formula C ₃₇ H ₃₈ O ₁₉ Na, is named kaempferol-3-O-(6′′-feruloyl)-sophoroside, and refers to a compound having the structure of [Formula 1] below do.

[Formula 1]

In the present invention, the compound may have muscle loss inhibitory activity, and specifically, may exhibit muscle loss inhibitory activity by inducing growth and differentiation of muscle cells, but is not limited thereto.

The compounds of the present invention can induce the growth and differentiation of muscle cells through the activation or down-regulation of specific signaling pathways, and preferably by activating the PI3K/Akt/mTOR signaling pathway or down-regulating the Smad signaling pathway. Growth and differentiation of muscle cells can be induced by, but is not limited thereto.

The compound of the present invention may be extracted and isolated from leek, but is not limited thereto.

As another aspect of the present invention, the present invention provides a pharmaceutical composition and health functional food composition for preventing, improving or treating sarcopenia, comprising the compound or a pharmaceutically or food-acceptable salt thereof as an active ingredient. .

As used herein, the term "prevention" refers to all activities to suppress or delay the onset of sarcopenia by administration of the composition according to the present invention.

The term "improvement" used in the present invention refers to all activities that reduce the degree of sarcopenia or alleviate symptoms such as complications caused by sarcopenia by administering the composition according to the present invention.

As used herein, the term "treatment" refers to all activities in which symptoms of sarcopenia are improved or beneficially changed by administration of the composition according to the present invention.

The novel compound represented by [Formula 1] according to the present invention may be used in the form of a salt. As the salt, acid addition salts formed by various organic or inorganic acids that are pharmaceutically or food-acceptable may be used. Acid addition salts are formed with inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanedios. It can be obtained from non-toxic organic acids such as oxalates, aromatic acids, aliphatic and aromatic sulfonic acids. These non-toxic salts include sulfate, pyrosulfate, bisulphate, sulphite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride. , acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propionic acid, oxalic acid, malonic acid, succinic acid, suberate, sebacate, fumarent, malic acid Eight, butyne-1,4-dioate, hexane-1,6-dioic acid, benzoic acid, chlorobenzoic acid, methylbenzoic acid, dinitrobenzoic acid, hydroxybenzoate, methoxybenzoic acid, pdalic acid, terepdalate, benzene Silphonic acid, toluenesulfonic acid, chlorobenzenesulfonic acid, xylenesulfonic acid, phenylacetic acid, phenylpropionic acid, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, It can be prepared using propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, trifluoroacetic acid and the like.

At this time, the acid addition salt according to the present invention is prepared by a conventional method, for example, dissolving the compound of Formula 1 in an excess acid aqueous solution, and adding the salt to a water-miscible organic solvent such as methanol, ethanol, acetone or It can be prepared by precipitation using acetonitrile. It can also be prepared by evaporating the solvent or excess acid from this mixture and then drying it, or by suction filtering the precipitated salt.

In addition, the novel compound represented by [Chemical Formula 1] according to the present invention can be made into a pharmaceutically or food-acceptable metal salt using a base. An alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is suitable for agrochemicals to prepare lithium, sodium, potassium or calcium salts as metal salts. In addition, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

The present inventors have identified the use of the novel compound according to the present invention for preventing, improving, and treating sarcopenia through specific examples.

Specifically, in one embodiment of the present invention, it was confirmed that the novel compound according to the present invention does not exhibit toxicity to C2C12 cells at a concentration of 50 μM and exhibits a skeletal muscle growth regulating effect by significantly increasing the differentiation of myoblast cells. (see Example 2).

In another embodiment of the present invention, the novel compound according to the present invention increases the expression levels of p-PI3K, p-Akt, p-FoxO1, p-mTOR and p-70S6K proteins, thereby increasing the PI3k/Akt/mTOR signaling pathway It was confirmed that growth of muscle cells could be induced by down-regulating the Smad signaling pathway by significantly reducing the protein expression levels of p-Smad2/3 and Smad4 (see Example 3).

Summarizing the results of the above examples, the novel compound according to the present invention is effective in improving sarcopenia by inducing the generation and differentiation of skeletal muscle, and can be widely used as pharmaceuticals, foods, and quasi-drugs for the prevention, improvement, and treatment of sarcopenia. It is hoped that you can

When the composition according to the present invention is in the form of a pharmaceutical composition, it may contain the compound of [Formula 1] alone or one or more pharmaceutically acceptable carriers in a pharmaceutically effective amount. At this time, the pharmaceutically acceptable carrier is one commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. In addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like may be further included.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically applied) depending on the desired method, and the dosage is dependent on the patient's condition, body weight, and disease. Depending on the degree, drug form, administration route and time, it can 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 with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, activity of the drug, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitantly used drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those 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, body weight, absorption rate, inactivation rate and excretion rate of the active ingredient in the body, type of disease, and concomitant drugs, generally 1 to 500 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 depending on the route of administration, gender, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

As another aspect of the present invention, the present invention provides a method for treating sarcopenia comprising administering the pharmaceutical composition to a subject.

In the present invention, the "individual" may be a mammal such as a rat, livestock, mouse, or human, and may specifically include a companion dog, a racehorse, or a human in need of anticancer treatment, preferably a human.

As another aspect of the present invention, the present invention provides a use of the pharmaceutical composition for the treatment of sarcopenia.

When the composition according to the present invention is used in the form of a health functional food composition, it can be prepared as a food with high medical and medical effects processed to efficiently display bioregulatory functions in addition to specific health food and nutrition supply, and the food Depending on the case, it can be mixed with functional food, health food, and health supplement food, and can be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. to obtain useful effects.

The health functional food of the present invention may include additional ingredients that are commonly used in food compositions to improve smell, taste, and vision. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu) may be included. In addition, amino acids such as lysine, tryptophan, cysteine, and valine may be included. In addition, preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dihydroacetate, etc.), disinfectants (bleaching powder, high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT) ), etc.), coloring agents (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite, etc.), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclemate, saccharin, sodium, etc.), Food additives such as flavoring agents (vanillin, lactones, etc.), expanding agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (thickeners), coating agents, gum base agents, foam inhibitors, solvents, improvers, etc. can be added. The additive may be selected according to the type of food and used in an appropriate amount.

When the health functional food of the present invention is used as a food additive, it may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods.

In the health functional food of the present invention, the content of the compound of [Formula 1] according to the present invention is not particularly limited, and may be variously changed depending on the condition of the subject to be administered, the type of specific disease, and the degree of progression. If necessary, it may also be included in the total content of food.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

[Example]

Example 1. Isolation and Experimental Methods of Novel Compounds

1-1. leek extract preparation

Chives native to Naju, Jeolla-do were purchased and used, and 4.0 kg of the above-ground part of the chives was dried at 40 ° C. for one day to obtain a dry weight of 450 g. The obtained dry weight was ultrasonically extracted with methanol at 30° C. for 3 hours each time, concentrated in a vacuum concentrator to remove the solvent, and 83.7 g of the concentrate was obtained.

1-2. Isolation of novel compounds from leek extract

After suspending 83.7 g of leek methanol extract obtained in Example 1-1 in water and fractionating with Hexane, CH ₂ Cl ₂ , and EtOAc using a separatory funnel, the remaining water fraction was concentrated under reduced pressure to obtain a total of 71.5 g of water. A fraction concentrate was obtained. The water fraction was divided into three small fractions by Diaion HP-20 column chromatography with 25%, 50%, and 75% methanol. Among them, the W2C fraction was CHCl ₃ -MeOH-Water (2:1:0.2) After silica gel column chromatography was performed, prep-HPLC was performed with 25% acetonitrile to isolate novel compound 1 (10 mg) represented by the following [Formula 1] (Yellow amorphous powder; C ₃₇ H ₃₈ O ₁₉ , HR-ESI -MS m/z: 809.1889 [M + Na]+ (calcd for C ₃₇ H ₃₈ O ₁₉ Na, 809.1900); for ¹ H (CD3OD, 400 MHz) and ¹³ C NMR (CD3OD, 100 MHz)).

[Formula 1]

1-3. Skeletal muscle cell culture

C2C12 cells, a mouse myoblasts cell line, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). In addition, DMEM, fetal bovine serum (FBS), horse serum (HS), penicillin-streptomycin, trypsin-EDTA and DPBS were purchased from Gibco-BRL (Burlington, Ont, Canada). C2C12 cells were maintained in DMEM containing 10% heat-inactivated FBS, streptomycin (100 mg/mL) and penicillin (100 unit/mL) at 37°C and 5% CO ₂ under humidified culture conditions. To induce differentiation, 80% confluent cultures were switched to DMEM containing 2% HS for 6 days with medium changes every other day.

1-4. Analysis of skeletal muscle cytotoxicity and cell proliferation activity

Cytotoxicity was evaluated by seeding the cells in 1 Х 10 ⁵ in a 96-well plate, treating the compound of [Formula 1] isolated in Example 1-2 for 24 hours after 24 hours, and evaluating cytotoxicity using the MTT assay. was performed. Formazan crystals were dissolved in DMSO, and absorbance was measured at 540 nm using an ELISA plate reader (BioTek Instruments, Inc., Winooski, VT, USA). The optical density of formazan produced in control cells not treated with the compound was considered to represent 100% viability.

BrdU assay (Millipore, Billerica, MA, USA) was used to evaluate the skeletal muscle cell proliferation activity. Cells were seeded with 5 Х 10 ⁴ in a 96-well plate, and 48 hours after seeding, the growth medium (DMEM containing 10% FBS) was replaced with differentiation medium (DMEM containing 2% HS) and performed every other day during the differentiation period. The compound isolated in Example 1-2 was treated. Finally, 6 days after differentiation induction, cell proliferation activity was measured using the BrdU assay.

1-5. Western blot analysis

Western blot analysis was performed to confirm the expression analysis of proteins related to the myogenesis signal transduction cascade of the novel compound according to the present invention. The novel compound according to the present invention was treated with a differentiation medium (DMEM containing 2% HS) during the cell differentiation period. Cytoplasmic and nuclear proteins were then collected and cellular proteins were separated by 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane. Membranes were blocked with 5% skim milk (in Tris-buffered saline containing 0.2% Tween-20) and incubated overnight at 4 °C with relevant primary antibodies (1:1000 dilution in 5% skim milk) to inhibit skeletal muscle cell synthesis or Activating proteins were analyzed. Next, HRP-conjugated secondary antibody (diluted 1:3000 in 5% skim milk) was added to the membrane and incubated for 90 minutes at room temperature. Bands were developed using a chemiluminescent substrate and photographed using the FUSION FX Spectra program (Vilber Lourmat, Marne-La-Vallιe, France) equipped with an eVo-6 camera. Band intensities were quantified using the Image J program.

Example 2. Confirmation of skeletal muscle cell differentiation activity of the novel compound according to the present invention

The result of measuring the cell viability of C2C12 myoblasts for the novel compound represented by [Formula 1] isolated in Example 1 by 3-(4-5-dimethyl-2yl)-2-5-diphynyltetrasolium bromide (MTT) analysis , As shown in FIG. 2a, the novel compound represented by [Formula 1] did not show a cytotoxic effect on C2C12 cells at a concentration of 50 μM.

In addition, the skeletal muscle cell differentiation activity of the novel compound represented by [Formula 1] was evaluated using a 5-bromo-2'-deoxyuridine (BrdU) cell proliferation assay. As a result, as shown in Figure 2b, it was confirmed that the novel compound according to the present invention significantly increased the differentiation of myoblasts compared to the control group. From the above results, it was found that the novel compound newly isolated from leek exhibits a skeletal muscle growth regulating effect.

Example 3. Confirmation of regulation of signal transduction pathway related to skeletal muscle growth by treatment with the novel compound of the present invention

3-1. Upregulation of the PI3K/Akt/mTOR signaling pathway

As disclosed in Examples 1-5, as a result of confirming the signal transduction pathway related to skeletal muscle growth through Western blot, as shown in FIGS. , significantly increased the protein expression levels of p-FoxO1, p-mTOR, p-70S6K and MyoD. That is, it can be inferred that the novel compound according to the present invention induces muscle cell growth through p-PI3K/Akt and p-mTOR/p70S6K.

3-2. Downregulation of the Smad signaling pathway

In order to analyze whether the novel compound according to the present invention regulates the Smad pathway, which is another signaling pathway related to muscle growth and differentiation, Western blot was performed to confirm the expression of p-Smad2/3 and Smad4 proteins related to the Smad signaling pathway. . As a result, as shown in Figures 4a to 4d, it was confirmed that the compound according to the present invention significantly reduced the protein expression levels of p-Smad2/3 and Smad4 compared to the control group.

From the above results, it can be seen that the novel compound according to the present invention activates the PI3K/Akt/mTOR signaling pathway and down-regulates the Smad pathway, thereby inhibiting the expression of MyoD, a nuclear factor involved in skeletal muscle development. It can be inferred that it has more influence. Therefore, it was found that the novel compound of [Formula 1] isolated from leek exhibits skeletal muscle growth and development effects by promoting the PI3K/Akt/mTOR pathway or down-regulating the Smad pathway.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

Claims (11)

A novel compound represented by Formula 1 below.
[Formula 1]

According to claim 1,
The compound is a novel compound, characterized in that it has a muscle loss inhibitory activity. According to claim 1,
The compound is a novel compound, characterized in that it induces the growth and differentiation of muscle cells. According to claim 1,
A novel compound, characterized in that the compound induces growth and differentiation of muscle cells by activating the PI3K/Akt/mTOR signaling pathway or downregulating the Smad signaling pathway. According to claim 1,
The compound is a novel compound, characterized in that extracted and isolated from leek. A pharmaceutical composition for preventing or treating sarcopenia, comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. According to claim 6,
The compound is a pharmaceutical composition for preventing or treating sarcopenia, characterized in that induces the growth and differentiation of muscle cells. According to claim 7,
The compound is a pharmaceutical composition for preventing or treating sarcopenia, characterized in that it induces growth and differentiation of muscle cells by activation of the PI3K / Akt / mTOR signaling pathway or downregulation of the Smad signaling pathway. A health functional food composition for preventing or improving sarcopenia, comprising the compound of claim 1 or a food chemically acceptable salt thereof as an active ingredient. According to claim 9,
The compound is a health functional food composition for preventing or improving sarcopenia, characterized in that it induces the growth and differentiation of muscle cells. According to claim 10,
The compound is a functional food composition for preventing or improving sarcopenia, characterized in that it induces growth and differentiation of muscle cells by activation of the PI3K / Akt / mTOR signaling pathway or downregulation of the Smad signaling pathway.

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