KR20190139521A - A Composition Comprising Extract of Enteromorpha for promoting muscle tissue production - Google Patents

Abstract

The present invention relates to a composition for promoting muscle tissue production containing an enteromorpha extract. When taking the enteromorpha extract of the present invention, an effect of promoting muscle tissue production is exhibited. The enteromorpha extract of the present invention has the effect of promoting muscle tissue production, it is suitable to be used as the composition for promoting muscle tissue production.

Description

A composition comprising extract of enteromorpha for promoting muscle tissue production}

The present invention relates to a composition for promoting muscle tissue production, including a green leaf extract.

The Republic of Korea is rapidly shifting to an aging age, and in 2017, the number of people aged 65 or over was 707 million, accounting for 13.8% of the total population. Recent studies have shown that as muscle age decreases, muscle tendency to decrease muscles tends to increase, and such decreases in muscle mass and muscle strength lead to weakness and weakness, and finally to independent activity. It makes life difficult and additionally causes physical disability. In addition, muscle reduction is recognized as a potential risk factor in an aging age because it can lead to adult disease and degenerative diseases of diabetes, obesity and hypertension.

On the other hand, green seaweed is a kind of green algae among seaweeds, and it is known to be rich in potassium and minerals as it is a kind of algae that is enjoyed in Korea and Japan, and the polysaccharides extracted from the seaweed have reduced blood lipid and antibacterial. , Anti-inflammatory and anticancer activity have been reported (Wang et al). Various studies have been conducted to isolate and purify the related green component to reveal its pharmacological action and mechanism of action.

Accordingly, an object of the present invention is to provide a composition for promoting muscle tissue production, including green extract.

The inventors of the present invention have found that the green extract can promote the production of muscle tissue to complete the present invention. Therefore, the problem solving means of the present invention is a composition for promoting the production of muscle tissue containing the green extract.

The green extract of the present invention has an effect of promoting the production of muscle tissue, it is suitable to be used as a composition for promoting muscle tissue production.

Figure 1 is a simplified diagram of the extraction process for producing the green extract of the present invention.
Figure 2 shows the cell activity measurement of the green distilled water extract used as a comparative example with the green extract of the present invention.
Figure 3 shows the results of the measurement of IGF-1 signaling pathway activation when treated with the green extract of the present invention.
Figure 4 shows the measurement results of the MEK signal pathway activation when treated with the green extract of the present invention.

The present invention provides a composition for promoting muscle tissue production, including the green extract.

The seaweed extract is 1) extracting the seaweed with distilled water;

2) separating the supernatant by centrifuging the extract;

3) adding 2-4 times the volume of ethanol to the supernatant to settle the sugar; And

4) It is obtained by a method comprising filtration of the settled sugar and then concentration of the filtrate under reduced pressure.

The green sea in the step 1) is preferably a green powder.

Distilled water in the step 1) is preferably used 20 to 30 times the weight of the green powder. If less than this, the active ingredient may not be extracted properly, if more than this there is a problem that the cost used for the entire extraction process increases.

Ethanol in step 3) is preferably used 2 to 4 times the volume of the supernatant. If less than this, the active ingredient may not be extracted properly, if more than this there is a problem that the cost used for the entire extraction process increases.

The extraction method of the present invention may further comprise 5) dissolving the obtained extract in distilled water to obtain a precipitate by adding ammonium sulfate.

The ammonium sulfate is commercially available for the purpose of precipitating the greenish extract, acetone may be used in addition to the ammonium sulfate.

The green extract of the present invention is suitable for being used as a composition for promoting muscle tissue production by showing the effect of promoting muscle tissue production.

The composition for promoting muscle tissue production of the present invention may further include other components having a muscle tissue production promoting effect in addition to the green extract.

The composition for promoting muscle tissue production of the present invention may include carriers, excipients and diluents that can be included in the drug in general, and the carriers, excipients and diluents that can be used include lactose, dextrose, sucrose, sorbitol, mannitol , Xylitol, Aceritol, Maltitol, Starch, Acacia Rubber, Alginate, Gelatin, Calcium Phosphate, Calcium Silicate, Cellulose, Methyl Cellulose, Microcrystalline Cellulose, Polyvinyrrolidone, Water, Methylhydroxybenzoate, Propylhydroxybenzo Eight, talc, magnesium stearate, and mineral oil are mentioned.

When formulating the composition for promoting muscle tissue production, it may include fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like that are generally used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. Or it may be prepared by mixing lactose (lactose), gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the composition for promoting muscle tissue production of the present invention can be used in combination with other conventional drugs used to treat muscle tissue damage. When used in combination with other conventional drugs, they may be administered sequentially or simultaneously, which can be readily determined by one skilled in the art.

The composition for promoting muscle tissue production of the present invention can be administered via any general route within the scope consistent with the object of the present invention. The composition of the present invention may be administered as desired, but is not limited to intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, pulmonary administration, rectal administration. The composition may also be administered by any device in which the active agent may migrate to the target cell.

Example

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited thereto.

Preparation of Laver Powder

Seagrass collected from the offshore of Busan was washed with clean water three to four times to remove salts and impurities, dried in a freeze dryer after washing, washing and dehydration, and then powdered with a food grinder to prepare a green powder.

Preparation of green extract of the present invention

5 L of distilled water and 200 g of green powder were immersed in a three-necked flask and extracted by stirring at room temperature for 4 hours. Thereafter, the supernatant was separated by centrifugation (3,000 rpm, 4 ° C., 30 minutes), and ethanol in three times the volume of the supernatant was added to react with sugar at low temperature for 24 hours. After filtration under reduced pressure to remove sugar, the filtrate was concentrated under reduced pressure. The residue obtained after centrifugation was extracted once more in the same manner and concentrated. The concentrate was dissolved in distilled water and protein was precipitated for 24 hours by adding ammonium sulfate and saturating. The precipitated protein was separated only by centrifugation (3,000 rpm, 4 ° C., 20 minutes), and stirred in distilled water for 48 hours using a dialysis membrane to remove ammonium sulfate remaining in the precipitated protein liquid. After that, finally obtained green extract was lyophilized and powdered. Figure 1 is a simplified process chart of the production process of green onion extract according to the present invention.

Comparative example

Preparation of Green Distilled Water Extract

In order to compare the cell activity with the extract of the present invention, the extract was prepared using only distilled water as the extraction solvent. First, 5 L of distilled water and 200 g of green powder were immersed in a three-necked flask, and extracted by stirring at room temperature for 4 hours. Thereafter, the supernatant was separated by centrifugation (3,000 rpm, 4 ° C., 30 minutes), and the obtained supernatant was lyophilized to be powdered and used for experiments.

Experimental Example 1. Evaluation of cell activity of the green extract of the present invention

In order to investigate the effect of promoting the muscle tissue production of the green extract according to the present invention, myoblasts (myoblast, skeletal muscle, L6 cells) were cultured by being sold in the ATCC (Americal Type Culture Collection). For the experiment, cells were cultured in 96 well plates at the level of 1 × 10 ⁶ cells / well, and DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS was used as a medium. After 24 hours of incubation in DMEM containing 10% FBS and about 60% of the surface area of the plate, serum free DMEM containing green sea extract prepared in Example and serum free DMEM containing green distilled water extract were incubated for 24 hours. . After 24 hours, the medium was removed to measure cellular activity and 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxiphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS, promega , USA) solution was added for 30 minutes and the absorbance was measured using an ELISA plate reader at 490 nm. The degree of muscle tissue promotion was calculated and expressed as a percentage based on the absorbance intensity of the control group not treated with the green extract. The results of measuring the cell activity are shown in FIG. 2, and the results of promoting cell activity in the case of the green leaf extract of the present invention were confirmed, but the distilled water extract extracted using only distilled water does not affect the cell activity. The point was confirmed.

Cell viability (%) = (absorbance of extract treated group / absorbance of control group) X100

Experimental Example 2. Analysis of the muscle tissue production promoting effect of the present invention green extract by Western blotting

In order to confirm the effect of promoting the muscle tissue production of the green extract of the present invention was carried out to confirm the cell growth promoting factor Insulin-like growth factor-1 (IGF-1) and its lower signaling. Myoblasts cultured on Ø100 plate were treated with RIPA Lysis buffer (2 μg / ml pepstatin, 2 g / ml Na ₃ VO ₄ , 2 μg / ml NaF, 1 μg / ml aprotinin, 5 μg / ml leupetin, 10 μg / ml phenylmethylsulphur) Homogenized in phenylfluoride) and centrifuged (12,000 rpm, 4 ℃, 20 minutes) to recover the supernatant. Protein concentration was measured with a BCA protein assay kit, and the protein amount was taken equally at 50 μg / lane, mixed with 5X sample buffer, boiled in a water bath and western blotted on 7.5%, 10% and 12.5% SDS-PAGE gels. Standard molecular weights were used with dual color markers and delivered to PVDF membranes (Millipore, USA). The membrane was blocked with 1% bovine serum albumin (BSA) / TBS-T, reacted at room temperature in the primary antibody for 2 hours, washed twice with TBS-T for 15 minutes, and then the secondary antibody was 1: 10,000. Dilution at -5,000 ratio was carried out for 1 hour. The reaction membrane was washed twice with TBS-T for 15 minutes, followed by fluorescent gel image analyzer using uper Signal West Pico Luminol / Enhancer solution and Super Signal West Pico Stable Peroxide solution (Rockford IL, USA). After developing, the protein bands were checked by scanning bands.

In this experimental example, the phosphorylation of IGF-1R of the IGF-1 pathway and the expression level of the lower signal pathway protein were confirmed, which are shown in FIGS. 3 and 4. As shown in FIG. 3, the phosphorylation of IGF-1R protein was increased in the green extract treatment group (EL-GP) compared to the control group, and the expression levels of shc and PY99, which are related proteins, were increased. It is believed that the green extract of the present invention has an effect on the metabolism, growth and differentiation of cells. As shown in FIG. 4, the expression of Ras / Raf protein and the phosphorylation of MEK and ERK proteins, which are induced by IGF to promote cell proliferation, were also increased in the green extract treatment group of the present invention compared to the control group. From this it is judged that the green extract of the present invention is involved in promoting the production of muscle tissue.

Claims (4)

1) extracting the seaweed with distilled water;
2) separating the supernatant by centrifuging the extract;
3) adding 2-4 times the volume of ethanol to the supernatant to settle the sugar; And
4) A composition for promoting muscle tissue production comprising a green leaf extract obtained by filtering the submerged sugar and then removing the filtrate under reduced pressure. The composition for promoting muscle tissue production according to claim 1, wherein the extraction method further comprises dissolving the obtained extract in distilled water and obtaining a precipitate by adding ammonium sulfate or acetone. The composition for promoting muscle tissue production according to claim 1, wherein the green onion in step 1) is a green powder. According to claim 1, wherein in step 3) ethanol is used as a three times volume of the supernatant composition for promoting muscle tissue production.

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