KR101771486B1 - The method for preparing panax ginseng extract with increased contents of selective dammaranes, and a pharmaceutical compositions of the same for prevention or treatment of sarcopenia-related diseases - Google Patents

Abstract

The present invention relates to a method for stimulating proliferation of muscle root cells containing at least one compound selected from the group consisting of 20 ( R ) ( S ) -gincenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3, Sarcopenia, myocardial infarction and cardiovascular diseases, and a process for producing the same. The ginsenoside is excellent in the action of activating the proliferation of muscle cells, and thus can be usefully used as a preventive or therapeutic agent for sarcopenia, myocardial infarction and cardiovascular diseases, and is useful for cosmetic, health food, animal feed, Can also be applied.

Description

TECHNICAL FIELD The present invention relates to a method for preparing ginseng extract and a composition for treating muscular dyspepsia-related diseases using the same, of sarcopenia-related diseases}

The present invention relates to a method for producing a ginseng extract in which the content of a specific ginsenoside which promotes regeneration of muscle cells is increased by using an enzyme treatment, a high temperature treatment or a high pressure treatment under an acidic condition.

In addition, the present invention relates to a method for preventing or treating sarcopenia, myocardial infarction and cardiovascular diseases caused by regression of muscle by activating the proliferation of muscle root cells, wherein the ginseng extract having an increased content of a specific ginsenoside is used .

Population aging is one of the most important social issues in the world. In the United States and Japan, the elderly population aged 65 and older is now expected to reach 13% and 16%, respectively, and 24% and 32%, respectively, after 25 years. In Japan, the current longevity-related industry in Japan is 39 trillion yen, which is expected to grow to 155 trillion yen in 20 years (2000, Japan Industrial Restructuring Review Committee, 2000). In Korea, the elderly population, which is 11.0% of the total population as of 2010, is expected to reach 24.3% by 2030. Therefore, urgent measures are needed.

One of the physiological changes that occur in the elderly is sarcopenia, which occurs with decreasing muscle mass as age increases. According to the Korean National Health and Nutrition Survey (IV), muscle mass began to decrease around 40 years for men and to decrease after 55 years for women (Hong S. et al., 2011). When defining the muscle thrombocytopenia in more than 65 years with ASM / height ^2, in one KLoSHA (Korean longitudinal study on health and aging) to target more than 65 years of community aged 565 people study man the limits of muscle thrombocytopenia was 7.09 kg / m ² and 5.27 kg / m ² in female, the prevalence of myopenia was reported to be 35.3% in male elderly and 13.4% in female elderly (Kim JH et al., 2010).

Even in the case of the general public, the concept of muscle-decreasing obesity, in which the body composition is changed and the body mass is decreased instead of increasing the body fat, is newly emerging even if the body weight does not change with age. In addition, lipid consumption and excessive nutrient intake lead to fat accumulation. In particular, intramuscular fat accumulation causes secretion of inflammatory cytokines such as TNF-alpha (tumor necrosis factor-alpha) and IL-6 (interleukin-6) This indirectly affects protein metabolism and directly affects insulin sensitivity, resulting in a decrease in muscle mass. Reduction of muscle mass has been reported to cause a vicious cycle that increases body fat by decreasing physical activity and reducing the basal metabolic rate, which is the caloric consumption of normal (Roubenoff R., 2000). These muscle weaknesses have also been reported to be associated with falls, trauma, dysfunction, increased hospitalization rates, decreased quality of life, and increased mortality due to decreased responsiveness to external stress due to muscle weakness JH et al., 2010).

Muscle hypoxia has been associated with various diseases and the incidence of myopenia in diabetic patients is three times higher than that of the general population, considering the age, sex, body mass index and lifestyle of the subjects in the diabetic patients, (Lim S. et al., 2010). In addition, another muscle in our body, the cardiac muscle (cardiac muscle) can also be a failure, a typical example is myocardial infarction. Myocardial infarction occurs when a thrombus formed in the coronary artery blocks the blood vessel and a part of the heart muscle is destroyed (necrosis). This myocardial infarction is one of the degenerative diseases, and the frequency of the onset is increasing as the aging society comes. Therefore, it is necessary to study the prevention and treatment considering various risk factors, and it is necessary to induce reduction of mortality due to myocardial infarction.

 The muscles of our body are divided into smooth muscle, cardiac muscle, and skeletal muscle. The skeletal muscle occupies a significant portion of our body, and promotes skeletal movement. These skeletal muscles do not divide, they are made up of polynuclear muscle fibers and are made during the embryo formation process. After the embryo formation process, the muscles are formed by postnatal growth or by the muscle neogenesis process. Especially when the muscles are damaged by frostbite, sprains, bruises, etc., muscular neoplasm occurs. In this process, satellite cells are activated and activated satellite cells are differentiated into myoblasts (Morgan J. E. et al., 2003). After differentiation, division of myofibroblasts occurs, fusion of myoblasts occurs and myotube develops. During these muscle neogenesis, when there are problems such as differentiation into myoblasts, division of myocytes, and various muscle disorders, a typical example is myopathy.

Recently, a method of regenerating muscle cells has been reported. Regeneration of such muscle cells is known to stimulate satellite cells existing outside the muscle cells, causing satellite cells to divide and form muscle tissue. Muscle cell regeneration has been reported to be applicable not only to damaged muscle repair but also to natural muscle loss due to aging (Conboy I. M. et al., 2003).

Recently, studies have been made to promote the differentiation of muscle cells using low molecular weight compounds. Recent studies in which substances promoting myogenesis by low molecular weight substances have been investigated include GSK3β inhibitors, calpain inhibitors and cAMP activators (cAMP activator). In fact, treatment with fibroblast growth factor (bFGF), forskolin, and the GSK3β inhibitor BIO has been shown to promote muscle cell differentiation (Xu C. et al., 2013). However, attempts to regenerate muscle using low molecular weight compounds have been made at the laboratory level, where toxicity evaluation and practical application have not been confirmed so far.

Panax ginseng (P anax ginseng CA Meyer) is a plant belonging to Araliaceae, and its roots are mainly used as medicinal parts. Especially in Korea, white ginseng (raw), red ginseng (red ginseng: steamed), and ginseng (蔘 蔘: thin roots) are used according to the medicinal effect. In the private sector, wild ginseng camphor and wild ginseng are distinguished. In China, it refers to the roots and rootstocks of ginseng and distinguishes it from raw ginseng (red ginseng), red ginseng and wild ginseng (wild ginseng). Ginseng has a peculiar odor, taste is slightly worn and it is known to be slightly warm (甘苦 微 温). It is used for body weakness, boredom, fatigue, anorexia, vomiting and diarrhea. And it is known that it increases the anxiety function and the new function. The pharmacological action of ginseng is widely used in the world as an important medicinal substance known to have various activities such as nourishing tonic action, immune enhancing action, central nervous system action, cardiovascular action and hypoglycemic action (Choi J. et al., 2013; Alraek T. et al., 2011).

In addition, there are various ginseng processed products such as ginseng radix rubra, black ginseng, fermented ginseng, and ginseng produced by the processing of ginseng. Examples of the same kind of ginseng include Panax quinquefolium , Panax japonicus , Panax notoginseng , and Panax stipuleanatus , Panax bipinnafidus , Panax zingiberensis , and Vietnamese panthera . In addition , ( Panax vietnamensis ) , and Panax sinensis .

The compounds present in ginseng are largely classified into protopanaxadiol (PPD), protopanaxatriol (PPT) ocotillol-type, and oleanolic acid (PPT) depending on the sugar- ), And various ginsenoside compounds have been isolated so far. Among the most known ginsenosides in terms of their effects and quantities, there are ginsenosides Rg2, Rg3, Rh1 and Rh2 which are abundant in processed ginseng. These ginsenosides are secondary ginsenosides produced by the hydrolysis of ginsenosides during processing of ginseng. These secondary ginsenosides have a number of novel activities. For example, ginsenosides Rh1, Rh2 and Rg3 have significant anticancer activities and induce regeneration of cancer cells (inducing cancer cells to regenerate into healthy cells) , And JINSEO NOSADEN Rg3 exhibit a relatively strong inhibitory effect on platelet aggregation induced by collagen and the like. Ginsenoside Rh1 has significant inhibitory effects on the conversion of thrombin-induced fibrinogen to fibrin.

The possibility of sarcopenia of ginseng extract was investigated by Cao et al. ( Caenorhabditis elegans ) in 2007. In this study, a gentle cell line expressing Myosin, a major protein of myofibrillar protein attached with green fluorescent protein, was prepared and administered ginkgo biloba extract and Guangdong ginseng extract, respectively. The increase in the amount of fluorescence expressed in the nematode administered with each extract suggests that the ginseng extract may be effective in myopenia (Cao Z. et al., 2007.).

The prior patents related to myopenia include "Use of Ginkgo biloba extract for the preparation of medicines for treating myopathy" (Korean Patent No. 0891393), Prevention and Treatment of Myopenia (Korean Patent Publication No. 2012-0058457) , Compositions for promoting muscle differentiation and muscle strengthening, and external preparations (Korea Patent Publication No. 2015-0024586). However, the above-mentioned patent does not disclose the treatment of muscle hypoxia caused by ginseng extract.

In addition, in the case of the prior literature related to ginseng extract processed at high temperature and high pressure containing various ginsenosides, a cosmetic composition for preventing skin aging (Korean Patent Publication No. 2010-0051405) containing "processed ginseng extract as an active ingredient" This patent relates to the anti-aging effect of the skin which is not related to the present patent. (Korean Patent No. 1348974) " and "Method for producing ginsenoside Rg3-fortified red ginseng extract and method for producing ginsenoside Rg3-enhanced red ginseng extract as an active ingredient (Korean Patent No. 1435930) differs from the ginseng fraction of the present invention in the extraction conditions and utilization purpose.

Therefore, the inventors of the present invention separated ginsenosides using ginseng and ginseng leaf extract to determine the optimal compound that can be used as a therapeutic agent for muscle hypoxia in ginseng, and measured the proliferative activity of each ginsenoside 20 ( R ) ( S ) -ginchenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3. Further, by developing a method for producing ginseng extract having an increased content of 20 ( R ) ( S ) -gincenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3, a new novel composition having a specific ginsenoside content increased It was confirmed that ginseng extract was used for myopenia, and thus it is effective to prevent or treat diseases related to senile muscle degeneration, myopenia, myocardial infarction and cardiovascular diseases, thus completing the present invention.

Korean Patent No. 0891393, Use of Ginkgo biloba extract for the manufacture of a medicament for the treatment of hypoxia, 2009.03.25. Enrollment. Korean Patent No. 1348974, Process for the production of new red ginseng by vacuum drying at high temperature and high pressure, 2014.01.02. Enrollment. Korean Patent No. 1435930, a method for producing ginsenoside Rg3-fortified red ginseng extract and a product containing the ginsenoside-enhanced red ginseng extract as an active ingredient by the method for preparing the same, 2014.08.25. Enrollment. Korean Patent Publication No. 2012-0058457, Prevention and Treatment of Muscle Reduction, June. open. Korean Unexamined Patent Publication No. 2010-0051405, a cosmetic composition for preventing aging of skin containing an extract of processed ginseng as an active ingredient, 2011.12.07. open. Korean Patent Laid-Open No. 2015-0024586, composition for promoting muscle differentiation and muscle strengthening, and external preparation, 2015.03.09. open.

In 2000, the Japan Industrial Restructuring Review Committee, "Report on the 21st Century Economic and Industrial Policy Vision Report, 2000. Alraek T. et al., Complementary and alternative medicine for patients with chronic fatigue syndrome: a systematic review. BMC Complement Altern. Med. 11 (87), 1-11, 2011. Choi J. et al., Ginseng for health care: a systematic review of randomized controlled trials in Korean literature. PLoS One. 8 (4). e59978. 2013. Conboy I. M. et al., Notch-mediated restoration of regenerative potential to aged muscle. Science, 302 (5650), 1575-1577, 2003. Cao Z. et. al., Ginkgo biloba extract EGb 761 and Wisconsin Ginseng delay sarcopenia in Caenorhabditis elegans. J Gerontola Biol Sci Med Sci., 62A (12), 1337-1345, 2007. Kim J. H. et al., Investigation of sarcopenia and its association with cardiometabolic risk factors in elderly subjects. J Korean Geriatr Soc., 14, 121-130, 2010. Lim S. et al., Sarcopenic obesity: prevalence and association with metabolic syndrome in the Korean Longitudinal Study of Health and Aging (KLoSHA). Diabetes Care, 33 (7), 1652-1654, 2010. Morgan J. E. et al., Muscle satellite cells. Int J Biochem Cell Biol., 35 (8), 1151-1156, 2003. Hong S. et al., Characteristics of body fat, body fat percentage and other body composition for Koreans from KNHANES IV. J Korean Med Sci., 26, 1599-1605, 2011. Roubenoff R., Sarcopenic obesity: does muscle loss cause fat gain? Lessons from rheumatoid arthritis and osteoarthritis. Ann NY Acad Sci., 904, 553-557,2000. Xu C. et al., A zebrafish embryo culture system defines factors that promotes vertebrate myogenesis across species. Cell, 155 (4), 909-921, 2013.

It is an object of the present invention to provide a method for producing a ginseng extract having an increased content of 20 ( R ) ( S ) -gincenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3.

The present invention also provides a composition which can be used for treatment of sarcopenia by promoting regeneration of source cells containing ginseng extract extracted by such a method.

It is a further object of the present invention to provide a ginseng extract having an increased content of 20 ( R ) ( S ) -gincenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3, sarcopenia), myocardial infarction and cardiovascular diseases, and a medicinal product, a health functional food or a functional food containing the extract.

The present invention relates to a pharmaceutical composition containing at least one ginsenoside selected from the group consisting of 20 ( R ) ( S ) -ginenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3 of the following formula 1, The present invention also relates to a composition for preventing or treating sarcopenia-associated diseases by promoting regeneration of muscle cells.

[Chemical Formula 1]

The ginseng extract is prepared by suspending ginseng in water, extracting ginseng with water, a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof, and then suspending the glucoside bond in the suspension And enzymes such as pectinase, hemicellulase, lactase, cellulase, etc., or a mixture of these enzymes, which are industrially used, It may be a concentrate obtained by pretreating with an enzyme used for removing sugar from a glycone-type compound attached to an existing sugar and then performing a heat treatment process for obtaining them.

In addition, medicines such as organic acids, acetic acid, hydrochloric acid, tartaric acid and the like can be used or acidic conditions such as omija, bokbunja, gugija, The extract of the ginseng fraction obtained by heat treatment at a high temperature and a high pressure, or a ginsenoside containing the ginsenoside isolated therefrom, activates the proliferation of sarcopenia (sarcopenia), myocardial infarction and cardiovascular A composition for preventing or treating a disease, and a process for producing the same.

The ginseng extract is pretreated with an acidic condition using a weak acid such as organic acid, acetic acid, hydrochloric acid, tartaric acid, malic acid, citric acid, lactic acid or the like, or a medicament such as omija, bokbunja, gugija, At this time, the optimal acidic condition for the enzyme reaction is pH 2 to 6, and the enzyme reaction at 35 to 70 ° C for 1 to 120 hours is 20 ( R ) ( S ) -ginchenoside Rh2, ginsenoside Rk2 and It is preferable to increase the content of ginsenoside Rh3.

When the ginseng extract obtained after the enzyme treatment is further subjected to the heat treatment at a high temperature and a high pressure under the conditions of a temperature of 100 to 125 ° C and a pressure of 1.0 to 100 atm for 30 minutes to 10 hours, 20 ( R ) ( S ) Rh2, ginsenoside Rk2 and ginsenoside Rh3.

The diseases to be treated by the activation of the muscle cells include, but are not limited to, muscle hypoxia, muscle hypoxia related aging, muscle tissue disorders, rhabdomyolysis, muscle cancer, myocardial infarction, cardiovascular diseases, metabolic diseases including diabetes mellitus, Can be selected.

The present invention also provides a pharmaceutical composition comprising at least one ginsenoside selected from the group consisting of 20 ( R ) ( S ) -ginchenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3 of Formula 1 isolated from the ginseng fraction And to a composition for the prophylaxis or treatment of diseases which are treated by activation of muscle cell proliferation.

In another aspect, the present invention is characterized in that it comprises at least one ginsenoside selected from the group consisting of 20 ( R ) ( S ) -ginnenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3 of the formula 1 Which is effective for preventing or ameliorating diseases treated by activation of muscle cell proliferation.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for producing ginseng by extracting and fractionating ginseng with water, a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof and then purifying and separating pure water using the chromatography and identifying the chemical structure and physicochemical properties The activity of the compositions of the present invention is confirmed by measuring whether the ginseng fraction and the compound represented by the formula (1) selectively proliferate the proliferation of human muscle root cells do.

The ginseng fraction of the present invention is selected as a composition for selectively activating proliferation of muscle root cells. Ginseng was selected by collecting various native plants and herbal medicines and measuring the activity of promoting proliferation of myocytes using C2C12 source cells derived from mammals. ( R ) ( S ) -ginsenoside Rh2, ginsenoside Rk2, and ginsenoside Rh3, respectively, as compared to the C2C12 source cells after the isolation of 56 kinds of ginsenosides from the ginseng extract, The proliferation of myofibroblasts was increased in the treated group. In order to confirm the selectivity of myofascial cell proliferation of compounds, representative of various types of cancer cells (HeLa, HCT116, YD-10B), mouse fibroblasts (3T3-NIH) and cancer-associated fibroblasts (CAF) It was confirmed that treatment with 20 ( R ) -ginsenoside Rh2, which is an active compound, selectively increased myofibroblast proliferation only. In addition, 20 ( R ) -ginsenoside Rh2 was harvested from the male gluteus maximus and cultured for primary source cells, and the degree of cell proliferation in human myocytes was confirmed.

For the exact mechanism of action of the compounds, the reduction of the cyclin dependent kinase inhibitors p57, p27 and p21 was confirmed in comparison to the untreated group treated with 20 ( R ) -ginnenoside Rh2. In addition, cell proliferation was observed in cardiomyocytes treated with 20 ( R ) -ginsenoside Rh2 as a result of examining whether these ginsenosides induce the proliferation of myocardial cells in addition to the source cells.

Therefore, natural substances derived from ginseng extracts have been shown to be effective in treating muscular dystrophy and muscular dystrophy related diseases.

When the ginseng fraction of the present invention is prepared, the ginseng fraction obtained by first concentrating ginseng with water, a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof is suspended in water After that, it is possible to break the glucoside bond in the suspension, and various enzymes used industrially, namely, β-glucosidase, pectinase, hemicellulase, lactase lactose, lipase, cellulase, glucose isomerase, xylanase, cellulose-1,4-beta-cellrobosidase (cellulose 1,4-beta -cellobiosidase, an enzyme such as alpha-rhamnosidase, esterase, and polygalacturonase, or a mixed composition thereof, The glycone Type enzymes used in removing the sugar from the compound of the present invention and then subjecting the result of the enzymatic reaction to heat treatment at a high temperature and a high pressure.

In addition, the ginseng extract can be pretreated with enzymes such as organic acids, acetic acid, hydrochloric acid, tartaric acid, etc., or by adding medicines such as omija and bokbunja which can produce organic acids, and then enzymatic reaction is performed. To obtain a concentrated ginseng extract.

In addition, water can be added to the concentrated ginseng extract after the enzyme treatment-heat treatment to prepare a suspension, followed by fractionation with various solvents. In this case, the extraction solvent for preparing ginseng extract is one time To 30 times the weight. The concentrated ginseng extract may be mixed with 1 to 10 L of water to prepare a suspension based on 0.0001 g to 10 g of the concentrated ginseng extract, followed by adding various solvents to prepare fractions. In addition, the solvent for fractionating the ginseng extract is preferably selected from hexane, ethyl acetate, or butanol. The solvent for the fraction is preferably 0.5 to 2 times the weight of the water in which the ginseng extract is suspended.

The ginsenosides of the present invention can be obtained by extracting ginseng extract or ginseng fraction with water, an organic solvent, an alcohol (methanol, ethanol, propanol) or a mixed solvent thereof, an organic solvent and water, Known methods used for the separation and extraction of the components can be easily obtained either singly or in suitable combination. The ginseng extract or fraction may be further purified according to a conventional method, if necessary. Preferably a ginseng extract or fraction; And purely separating and purifying a compound having an effect of proliferating muscle cells by using chromatography.

Examples of the chromatography used in the present invention include silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, thin layer chromatography Thin layer chromatography (TLC) and high performance liquid chromatography may be used.

The Panax ginseng fraction or the ginsenoside isolated therefrom acts on the proliferation of muscle cells and thus is useful for the treatment of diseases associated with muscle cell depletion such as aging, muscle tissue disorders, metabolic diseases including diabetes, ataxia, The composition may be used as a composition for preventing or treating various diseases consisting of muscular dystrophy, preferably a composition for preventing or treating muscular dysgenesis.

The ginseng fraction of the present invention or the ginsenoside isolated therefrom can be easily separated from ginseng and can be used as an additive for foods and pharmaceuticals since its stability is high.

The present invention provides a pharmaceutical composition for preventing or treating diseases which are treated by selectively activating the proliferation of muscle cells containing ginseng fraction or ginsenoside isolated therefrom. The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient in the ginseng fraction of the present invention or the ginsenosides isolated therefrom, such as starch , Calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The dosage of the ginseng fraction of the present invention or the pharmaceutical composition containing the ginsenoside separated therefrom depends on the age, sex, weight and the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, It will depend on the judgment of the person. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The ginseng fraction of the present invention or a pharmaceutical composition containing the ginsenoside isolated therefrom can be administered to mammals such as rats, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Since the extract of the present invention has little toxicity and side effects, it can be safely used even for long-term administration for the purpose of prevention.

The present invention also provides a health functional food comprising a ginseng fraction or a food-acceptable food-aid additive containing ginsenosides isolated therefrom. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamins , And health functional foods. In particular, the present invention relates to a method for the prevention or amelioration of a disease to be treated by selectively activating the proliferation of muscle root cells comprising the ginseng fraction or a pharmaceutically acceptable food supplement containing the ginsenoside isolated therefrom, Provide functional foods.

The present invention relates to a pharmaceutical composition comprising a composition containing Panax ginseng extract or ginsenoside isolated therefrom, which selectively activates proliferation of myofascial cells, and is useful as a medicament for the treatment of myopenia, myocardial infarction and cardiovascular A medicament for preventing or treating disease, a health food, or a functional food.

Figure 1 shows the results of comparing the amounts of ginsenosides Rd, F2 and 20 ( R ) ( S ) -ginnenoside Rh2 produced after treatment of various enzymes to obtain protopanaxidiol series of ginsenosides. Fig. 1 (A) shows the result of treatment with cellulolytic enzyme. Fig. 1 (B) shows the result of treatment with biscozyme enzyme and Fig. 1 (C) shows the result of treatment with beta-glucosidase enzyme.
FIG. 2 shows the results of comparing the amounts of ginsenosides Rd, F2 and 20 ( R ) ( S ) -ginnenoside Rh2 produced by activation of viscose, an immobilized enzyme. FIG. 2 (A) shows the result of checking the amount of Rd, F2 and 20 ( R ) ( S ) -ginnenoside Rh2 produced according to the amount of the enzyme treated. FIG. The results of comparing the amounts of ginsenosides Rd, F2 and 20 ( R ) ( S ) -ginsenoside Rh2 produced.
FIG. 3 shows the result of analyzing compounds produced by treating mushnosides Rd, F2 and (S) -gincenoside Rg3 with purified water, and then treating them with high temperature and high pressure.
Fig. 4 shows the results that the ginsenosides Rd and F2 are decreased in proportion to the heat treatment time and the aimed 20 ( R ), ( S ) -ginnenoside Rh2 is increased.
Figure 5 shows the results of a ginsenoside-derived ginsenoside screened using a cell-based screening system. The degree of cell proliferation was evaluated using the MTT assay. Y axis represents the degree of cell proliferation compared to the control group in%.
FIG. 6 shows the results of measuring the degree of proliferation of C2C12 myocytes by the WST-1 test by treating 20 ( R ) -ginsenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3, respectively.
FIG. 7 shows the result of measuring the degree of cell proliferation by treating 20 ( R ) -ginsenoside Rh2 with a fibroblast cell line.
FIG. 8 is a graph showing the effect of BrdU (5-bromo-2'-deoxyuridine) on mouse-derived stem cell C2C12 (A), male glutathione stem cell (B), and cardiomyocyte C). BIO was used as a control.
Figure 9 shows mRNA and protein expression levels of the cyclin dependent kinase inhibitors p57, p21, and p27, which affect cell cycle in C2C12 myocytes treated with 20 ( R ) -ginsenoside Rh2 to be. FIG. 9 (A) shows the result of confirming mRNA expression levels of p57, p21 and p27 by RT-PCR, and GAPDH was used as a loading control to confirm that all samples contained the same amount of mRNA. Figure 9 (B) shows the results of western blotting of p57, p27 and p21 protein expression levels. Alpha-tobulline was used as a loading control to confirm that all samples contained the same amount of protein.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

≪ Example 1: Preparation of ginseng extract >

20 g of ground ginseng was placed in 400 ml of each solvent (see Table 1), and the active material was extracted once or three times for 2 hours using an ultrasonic wave extractor.

Ginseng components can be extracted with organic solvents such as water, alcohol, and ether. When extracting ginseng, fatty acids, various hydrocarbons, polyacetylene compounds, steroids and terpenoids present in ginseng are relatively low, In order to obtain sugar and ginsenoside glycosides, as shown in Table 1, it was confirmed that extracting the components using water was optimal, and the total ginsenoside content was measured to be 98.6 to 118.7 mg per g of 20 g of ginseng Respectively.

To determine the content of 20 ( R ) ( S ) -ginsenoside Rh2, ginsenosides Rk2 and Rh3, which promote the proliferation of muscle cells in the water extract of ginseng, aqueous methanol solution (63 → 100% [v / v]) High performance liquid chromatography (Optima Pak C ₁₈ column, 4.6 x 250 mm, particle size 5 mu m, flow rate 1 ml / min, UV detection: 260 nm) was performed according to concentration gradient elution conditions. As a result, 20 ( R ) ( S ) -ginsenoside Rh2, ginsenosides Rk2 and Rh3 were not detected.

Extraction solvent Comparing the extraction amount of ginseng ingredients Water extract 35.3% 10% aqueous solution of ethanol 36.1% 30% ethanol aqueous solution extract 33.3% 70% ethanol aqueous solution extract 22.6% 100% ethanol aqueous solution extract * 18.2% 100% aqueous methanol solution extract * 15.4% Ether extract * 0.84% Ethyl acetate extract * 8.45% * Add the same amount of solvent, extract three times with an ultrasonic extractor, and concentrate.

Example 2: Separation of ginsenoside of protopanaxidiol series from ginseng extract < RTI ID = 0.0 >

Among the components of ginseng, HP-20 column chromatography was used for optimization and concentration of the ginsenoside content of the protopanaxdiol system.

The ginseng water extract was fractionated by divalent anionic HP-20 column chromatography.

Confirmation of the content of 20 ( R ) ( S ) -ginsenoside Rh2, ginsenoside Rk2 and Rh3 present in the fraction containing the ginsenoside of the protopanaxidiol system fractionated by HP-20 column chromatography High performance liquid chromatography (Optima Pak C ₁₈ column, 4.6 x 250 mm, 5 μm particle size, flow rate 1 ml / min) according to concentration gradient elution conditions of methanol aqueous solution (63 → 100% [v / , UV detection: 260 nm]. As a result, 20 ( R ) ( S ) -ginsenoside Rh2, ginsenosides Rk2 and Rh3 were not detected.

&Lt; Example 3: Enzyme treatment conditions >

Example 3-1. Comparison of enzyme activity

Generally, enzymes can break the glucoside bond and can be used in various enzymes used industrially such as beta-glucosidase, pectinase, hemicellulase, lactase, cellulase ), Or a mixed composition thereof can be used. Table 2 summarizes the enzymes used in the present invention and their enzymes.

    The pH value of the ginseng fraction obtained through HP-20 column chromatography was measured to be 3.0 to 4.5. To these fractions, 1 to 10% by weight of an enzyme solution (see Table 2) was added to the reaction mixture, and the mixture was stirred at 37 DEG C for 1 hour to 72 hours for enzyme reaction. At this time, to control the pH (pH 3 to 6) of the enzyme to be used, an extract of herbal medicine such as Omija was treated in the same manner.

After high-performance liquid chromatography [Optima Pak C ₁₈ column, 4.6 x (v / v)] according to the concentration gradient elution condition of aqueous methanol solution (63 → 100% [v / v]) to confirm the component change of the ginsenoside after the enzyme reaction 250 탆, 5 탆 particle size, flow rate 1 ml / min, UV detection: 260 nm]. The results of analysis of the compositional change of Jinsen-no-sai by each enzyme treatment are shown in Table 2 and Fig.

Enzyme used Characteristics of enzymes Changes in ginsenosides Ultrazyme 100G Pectinase Trace increase of Rg3 or Rh2 Novozyme 435 Immobilized lipase Trace Rg3 increase Novozyme 33095 Pectinase Produced Compound K Lecitase Ultra lipase Production of trace Rg3, Compound K Pectimex Ultra AFP Robust pectinase / hemicellulose complex Trace increase of Rg3 or Rh2 Sweetzyme Extra IT Immobilized glucose isomerase No change Novarom Blanc beta-glucosidase Produced Compound K Viscozyme Carbohydrase Cellulase, xylanase ... etc Produced Gin Senocide Rd, F2 Celluclast Cellulase, cellulose 1,4-beta-cellobiosidase Produced Gin Senocide Rd

As shown in the results of Table 2, Novozyme 33095 (Novozyme 33095), Lecitase Ultra AFP and Novarom Blanc produced a large amount of compound K specifically, Looking at Table 2 and FIG. 1 (A), Celluclast increased the production of ginsenoside Rd. It was also observed that the production of ginsenosides Rd and F2 increased with the reaction time when the viscose of Fig. 1 (B) and the beta-glucosidase of Fig. 1 (C) were treated. The results of Table 2 and FIG. 1 indicate that viscose was selected to optimize the increase of ginsenoside 20 ( R ) ( S ) -ginsenoside Rh2, ginsenoside Rk2 and Rh3 compounds.

Example 3-2. Immobilization of enzyme

When the liquefied enzyme is treated in the extract of ginseng, the cost of the enzyme and the enzyme removal process after using the enzyme are needed. In order to overcome this problem, we have developed a method for immobilization of enzymes for the reaction. Agar was used as a method of fixing the enzyme, but the present invention is not limited thereto.

15 g of agar was added to 150 ml of distilled water to make a 10% agar solution, sterilized using a high pressure autoclave, and the agar solution was cooled to 50 to 60 캜. The enzyme to be used was added to the cooled agar solution, which was then cooled and immobilized as a gel. In this case, the enzyme was viscose.

To confirm the reactivity and reuse of the immobilized enzyme solution, ginsenosides Rd and F2 were analyzed. The enzyme treatment was carried out in the same manner as in Example 3-1, except that the immobilized enzyme solution was stacked on a cotton or iron mesh to prevent the enzyme solution from being mixed with the ginseng extract or crushed. In order to confirm the reuse of the immobilized enzyme solution, the immobilized enzyme solution used in the reaction was repeated three times by adding a new ginseng extract to the solution. The results are shown in Fig.

As shown in FIG. 2 (A), it was confirmed that the amount of ginsenoside Rd and F2 increased with the addition amount of the immobilized biscochrome enzyme. In addition, as shown in FIG. 2 (B), when the immobilized enzyme solution was used three times repeatedly, it was found that the enzyme activity was maintained at the first two-thirds.

Thus, when the immobilized enzyme solution is used, the reaction activity of the enzyme can be maintained, and it is advantageous that the enzyme can be used in a large number of regeneration. Further, side effects and additional reaction steps are required depending on the presence or absence of the industrial enzyme present in the ginseng extract. However, if the immobilized enzyme of the present invention is used, such a problem can be fundamentally eliminated.

Example  3-3. Content Increased Protopanaxidiol  Family Gin Senocide  Enzyme treatment to obtain

The immobilized biscozyme enzyme prepared in Example 3-2 was added to the ginseng fraction obtained through HP-20 column chromatography, and the mixture was stirred at 37 ° C for 72 hours for enzyme reaction.

< Example  4: heat treatment>

Example  4-1. Heat treatment Gin Senocide  Confirm

The ginsenosides Rd, F2 and (S) -ginsenoside Rg3 were subjected to high-temperature and high-pressure treatment in order to confirm the substances produced when the ginsenosides were heat-treated.

The purified product was dissolved in 2 ml of distilled water using 1 mg of each of purified ginsenoside Rd, F2 and (S) -ginchenoid Rg3, followed by repeated heat treatment three times in a high pressure sterilizer for 2 hours, Respectively. The HPLC analysis was carried out in the same manner as in Example 1 above.

As shown in FIG. 3, when the ginsenoside Rd was heat-treated, ( R ), ( S ) -ginenoside Rg3, ginsenosides Rk1 and Rg5 were produced specifically (Rd reference AuC), (S) - Gene, if the heat treatment the ginsenoside Rg3, the (R) - ginsenoside Rg3, ginsenoside Rk1, Rg5, 20 (R) , (S) - ginsenoside Rh2, ginsenoside Rk2 and Rh3 were generated (see Rg3 AuC). Further, 20 ( R ), ( S ) -ginenoside Rh2, ginsenosides Rk2 and Rh3 were produced as target products (see F2 AuC) by heat treatment of ginsenoside F2.

In order to produce 20 ( R ), ( S ) -ginenoside Rh2, ginsenoside Rk2 and Rh3, which are the object of the present invention, the herbal medicine such as enzyme and omija are added to the water extract of ginseng, The extracts were then heat-treated again.

Example  4-2. purpose Gin Senocide  Heat treatment to obtain

The enzyme-treated ginseng extract obtained from Example 3-3 was heat-treated for 3 hours in a high-pressure sterilizer at 120 占 폚 for 2 hours. After the heat treatment, ginsenoside 20 ( R ), ( S ) -ginsenoside Rh2, ginsenoside Rk2 and Rh3 were isolated and purified.

As shown in FIG. 4, when the ginsenosides Rd and F2 were heat-treated, 20 ( R ), ( S ) -ginnenoside Rh2 was produced in proportion to the heat treatment time.

&Lt; Example 5: Preparation of 20 ( R ), ( S ) - Physicochemical Properties and Structure Analysis of Ginsenoside Rh2, Ginsenoside Rk2 and Rh3>

Example 5-1. 20 ( R ) - ginsenoside Rh ₂ ,

ESI-MS m / z : 645 [M + Na] &lt; ^{+ &}

¹ H-NMR (500 MHz, d ₅ -pyridine): 1.49 (1H, m, H-1a), 0.79 , m, H-2b), 3.40 (1H, dd, J = 11.5, 3.7 Hz, H-3), 0.73 (1H, br d, J = 11.5 Hz, H-5), 1.50 (2H, m, H (1H, m, H-11a), 1.13 (1H, m, H-7) (1H, m, H-11b), 2.0 (1H, m, H-13) M, H-16b), 1.91 (1H, m, H-16a), 1.35 m, H-23), 2.45 (1H, m, H-23), 1.42 (3H, s, ), 5.34 (1H, t-like, H-24), 1.72 (3H, s, H-26), 1.68 3H, s, H-29), 0.98 (3H, s, H-30); 3-Glc: 4.98 (1H, d, J = 7.6 Hz, H-1 '), 4.02 (1H, m, H-2'), 4.22 (1H, m, H-3 '), 4.18 (1H, m , H-4 '), 3.98 (1H, m, H-5'), 4.57 (1H, d, J = 11.9 Hz, H-6'a), 4.37 (1H, dd, J = 11.9, 5.5 Hz, H-6'b).

^{13 C-NMR (125 Hz,} d 5 -pyridine): 39.6 (C-1), 27.2 (C-2), 89.2 (C-3), 40.5 (C-4), 56.8 (C-5), 18.9 (C-6), 35.6 (C-7), 37.4 (C-8), 50.8 (C-9), 40.1 C-13), 52.2 (C-14), 31.8 (C-15), 27.1 (C-16), 51.1 C-23), 23.2 (C-23), 126.5 (C-24), 131.2 (C-25), 26.3 27), 28.6 (C-28), 16.3 (C-29), 17.8 (C-30); C-3 '), 72.3 (C-4'), 78.8 (C-5 '), 63.5 (C-6' ).

Example 5-2. 20 ( S ) - ginsenoside Rh ₂

ESI-MS m / z : 645 [M + Na] &lt; ^{+ &}

¹ H-NMR (500 MHz, d ₅ -pyridine): 1.49 (1H, m, H-1a), 0.79 (m, H-2b), 3.40 (1H, dd, J = 11.6, 4.0 Hz, H-3), 0.73 (1H, d, J = 11.6 Hz, H- (1H, m, H-11), 1.12 (1H, m, H-7) m, H-11b), 4.10 (1H, m, H-12), 2.01 (1H, m, H-13), 2.01 ), 1.88 (1H, m, H-16a), 1.36 (1H, m, H-16b), 2.35 m, H-23a), 1.44 (3H, s, H-21), 2.01 , 2.30 (1H, m, H-23b), 5.33 (1H, t-like, H-24), 1.70 , s, H-28), 0.97 (3H, s, H-29), 0.95 (3H, s, H-30); 3-Glc: 4.96 (1H, d, J = 7.6 Hz, H-1 '), 4.02 (1H, m, H-2'), 4.23 (1H, m, H-3 '), 4.18 (1H, m , H-4 '), 3.91 (1H, m, H-5'), 4.56 (1H, d, J = 11.9 Hz, H-6'a), 3.77 (1H, dd, J = 11.9, 5.5 Hz, H-6'b).

^{13 C-NMR (125 Hz,} d 5 -pyridine): 39.6 (C-1), 27.5 (C-2), 89.2 (C-3), 40.5 (C-4), 56.8 (C-5), 18.9 (C-6), 35.6 (C-7), 37.4 (C-8), 50.8 (C-9), 40.1 C-13), 52.2 (C-14), 31.8 (C-15), 27.2 (C-16), 55.2 C-26), 18.2 (C-22), 27.3 (C-21), 36.3 27), 28.6 (C-28), 16.3 (C-29), 17.5 (C-30); C-3 '), 72.3 (C-4'), 78.8 (C-5 '), 63.5 (C-6' ).

Example 5-3. Gin Senocide Rk ₂

ESI-MS m / z : 627 [M + Na] &lt; ^{+ &}

¹ H-NMR (500 MHz, d ₅ -pyridine): 1.49 (1H, m, H-1a), 0.75 , m, H-2b), 3.40 (1H, dd, J = 11.5, 4.2 Hz, H-3), 0.77 (1H, d, J = 10.5 Hz, H-5), 1.47 (1H, m, H- (1H, m, H-9), 1.91 (1H, m, H-7) m, H-11a), 1.40 (1H, m, H-11b), 3.95 (1H, m, H-12), 2.01 ), 1.06 (1H, m, H-15b), 2.06 (1H, m, H-16a), 1.57 (1H, s, H-18), 0.82 (3H, s, H-19), 5.17 , 2.38 (1H, m, H-22b), 2.32 (1H, m, H-23), 5.30 , s, H-27), 1.34 (3H, s, H-28), 1.02 (3H, s, H-29), 0.99 (3H, s, H-30); 3-Glc: 4.96 (1H, d, J = 7.7 Hz, H-1 '), 4.01 (1H, m, H-2'), 4.20 (1H, m, H-3 '), 4.18 (1H, m , H-4 '), 3.91 (1H, m, H-5'), 4.56 (1H, d, J = 11.8 Hz, H-6'a), 3.77 (1H, dd, J = 11.9, 5.5 Hz, H-6'b).

^{13 C-NMR (125 Hz,} d 5 -pyridine): 39.1 (C-1), 28.1 (C-2), 89.2 (C-3), 40.5 (C-4), 56.3 (C-5), 18.4 (C-6), 35.2 (C-7), 39.6 (C-8), 50.6 (C-9), 37.2 C-13), 50.8 (C-14), 32.6 (C-15), 30.9 (C-16), 48.4 C-24), 131.6 (C-25), 25.9 (C-26), 17.9 (C- 27), 28.8 (C-28), 16.9 (C-29), 17.0 (C-30); (C-1 '), 75.9 (C-2'), 78.7 (C-3 '), 71.8 ).

Example 5-4. Ginsenoside Rh ₃

ESI-MS m / z : 627 [M + Na] &lt; ^{+ &}

¹ H-NMR (500 MHz, d ₅ -pyridine): 1.47 (1H, m, H-1a), 0.75 , m, H-2b), 3.40 (1H, dd, J = 11.5, 4.1 Hz, H-3), 0.74 (1H, d, J = 10.5 Hz, H-5), 1.49 (1H, m, H- M, H-9), 1.91 (1H, m, H-7), 1.36 m, H-11a), 1.41 (1H, m, H-11b), 3.95 M, H-16b), 1.98 (1H, m, H-17), 1.02 (3H, s, H-18), 0.81 (3H, s, H-19), 1.83 (3H, s, H-21), 5.52 (1H, t, J = 6.1 Hz, H-22), 2.38 (1H, m H-22b), 2.71 (2H, m, H-23), 5.24 (1H, t, J = 7.0Hz, H- H-27), 1.20 (3H, s, H-28), 1.10 (3H, s, H-29), 0.95 (3H, s, H-30); 3-Glc: 4.96 (1H, d, J = 7.6 Hz, H-1 '), 4.02 (1H, m, H-2'), 4.21 (1H, m, H-3 '), 4.19 (1H, m , H-4 '), 3.91 (1H, m, H-5'), 4.56 (1H, d, J = 11.7 Hz, H-6'a), 3.76 (1H, dd, J = 11.8, 5.4 Hz, H-6'b).

^{13 C-NMR (125 Hz,} d 5 -pyridine): 39.1 (C-1), 28.1 (C-2), 89.4 (C-3), 40.5 (C-4), 56.3 (C-5), 18.4 (C-6), 35.2 (C-7), 39.6 (C-8), 50.6 (C-9), 37.2 C-13), 50.8 (C-14), 32.6 (C-15), 26.6 (C-16), 50.8 C-23), 123.8 (C-24), 131.2 (C-25), 25.7 (C-26), 17.7 (C- 27), 28.8 (C-28), 16.9 (C-29), 17.0 (C-30); (C-3 '), 71.8 (C-4'), 78.3 (C-5 '), 63.1 ).

&Lt; Example 6: Preparation of 20 ( R ), ( S ) - Establishment of mass production process of ginsenoside Rh2, ginsenoside Rk2 and Rh3>

Ginsenoside 20 ( R ), ( S ) -ginsenoside Rh2, ginsenoside Rk2 and Rh3 were steamed.

Hot water was added to the ginseng and the active material was extracted with an ultrasonic wave extractor once or three times for 2 hours. The extract of ginseng was fractionated by column chromatography on artificial ion HP-20 to obtain protopanaxidiol-based ginsenosides. The biscozyme enzyme immobilized on the ginseng extract obtained from HP-20 column chromatography was added thereto, and the mixture was stirred at 37 ° C for 72 hours for enzymatic reaction. At this time, one of organic acid, lactic acid, and omiza was selected and processed together to adjust the pH of the enzyme. The enzyme - treated ginseng extract was heat - treated for 3 hours in a high - pressure sterilizer for 2 hours.

<Example 7: Cell proliferation inducing activity of ginsenoside derived from ginseng extract>

A cell - based screening system was used to confirm the cell proliferation - inducing activity of ginsenoside - derived ginsenoside. The cell proliferation inducing activity was measured by MTT assay.

C2C12 mouse stem cells were cultured in growth medium consisting of DMEM supplemented with 10% fetal bovine serum (Gibco), 50 U / mL penicillin and 50 μg / mL streptomycin (Dulbecco's Modified Eagle's Medium; Invitrogen, OR, USA) Respectively. The incubator used for cell culture was maintained at a temperature of 37 ° C and a CO ₂ concentration of 5%.

C2C12 cells cultured to perform the MTT assay were dispensed into a 96-well tissue culture plate (BD Falcon, NJ, USA) at a concentration of 3000 cells / well and cultured for 24 hours. After 24 hours, ginsenoside derived from ginseng extract was treated to a concentration of 10 占 퐂 / ml and then cultured for 48 hours. After 48 hours, 200 占 퐇 / well of the MTT solution diluted in the serum-free medium was added to the cells. After incubation for 2 hours in a CO ₂ incubator, the medium was removed, and 50 μl of DMSO was added. The mixture was reacted for about 10 minutes in an agitator, and then eluted using an ELISA (VERSA Max microplate reader, Molecular Devices, CA, USA) Absorbance was measured at a wavelength of ㎚.

As a result, as shown in Fig. 5, 20 ( R ) -ginsenoside Rh2 (531), ginsenoside Rk2 (533) and ginsenoside Rh3 (534) among ginsenoside- The highest proliferation activity was observed.

&Lt; Example 8: Preparation of 20 ( R ) -Induction of myosinide Rh2, ginsenoside Rk2 and ginsenoside Rh3 in myelin proliferation>

The cultured C2C12 cells were seeded on 96-well tissue culture plates at a concentration of 5000 cells / well and cultured for 24 hours. After 24 hours, the 20 ( R ) -ginsenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3 were sufficiently dissolved in the cell culture medium, and then treated with the culture medium in each well containing the cells. After 48 hours of treatment, WST-1 (Hoffmann-La Roche Switzerland) was treated with 10 μl of each well, and the absorbance was measured at 450 nm wavelength using ELISA (VERSA max microplate reader USA) two hours later. The results are shown in Fig.

As shown in FIG. 6, a group treated with 5 μg / ml of 20 ( R ) -ginnenoside Rh2, 10, 5, 2.5 μg / ml of ginsenoside Rk2 and 10, 5 μg / ml of ginsenoside Rh3 Were significantly different from those in the control group.

&Lt; Example 9: Identification of cell specific activity of ginsenoside >

In order to determine whether 20 ( R ) -gincenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3 induce cell proliferation in cells other than myoblast cells, 3T3-NIH, a mouse early fibroblast, and CAF, MTT assays were performed using HCT116 (human colorectal cancer cells), YD-10B (human oral cancer cells) and HeLa (human cervical cancer cells) cells.

The MTT test was carried out in the same manner as in Example 7 except that only 20 ( R ) -ginnenoside Rh2 in ginsenoside was used. At this time, 20 ( R ) -ginsenoside Rh2 was treated so as to have a final concentration of 5 占 퐂 / ml or 10 占 퐂 / ml.

As shown in Fig. 7, 20 ( R ) -ginsenoside Rh2 reduced the proliferation of cancer-associated fibroblasts and did not affect the proliferation of mouse fibroblasts and human cancer cell lines tested. This means that 20 ( R ) -ginsenoside Rh2 does not proliferate fibroblasts or cancer cells capable of specifically proliferating source cells and forming scars.

&Lt; Example 10: Confirmation of cell proliferation using BrdU immunostaining >

To confirm that 20 ( R ) -ginsenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3 induce proliferation of myoblast cells, BrdU (5-Bromo-2'-deoxyuridine, 5-bromo- Deoxyuridine) immunostaining was used. BrdU acts as an analog of tymidine, the DNA base, which can intercalate between DNA bases during DNA replication. At this time, the degree of cell proliferation can be confirmed by detecting BrdU-positive cells using an antibody specifically acting on BrdU. 20 ( R ) -ginsenoside Rh2 was used as the ginsenoside, and BIO (GSK3? Inhibitor), which is one of the substances promoting muscle differentiation, was used as a control group to compare the activity. Cells were obtained from C2C12 cells, the source cells of the mice, myocytes from the male gluteus, and cardiomyocytes isolated from the newborn mice.

Each of the cells was divided into 6-well tissue culture plates (BD Falcon, NJ, USA) at a concentration of 10000 cells / well and cultured for 24 hours. Twenty-four hours later, 20 ( R ) -ginsenoside-derived Rh2 from ginseng extract was adjusted to a concentration of 5 占 퐂 / ml or 10 占 퐂 / ml, and the BIO was treated to be 5 uM and cultured for 72 hours in total. Cells were treated with 10 uM BrdU (Sigma-aldrich, SL, USA) for 48 hours after treatment with ginsenoside, and the cells were further cultured for 24 hours. Then, the medium was removed and the cells were treated with phosphate buffered saline The medium was removed and 3.4% formaldehyde (Sigma-Aldrich, SL, USA) (1 ml) diluted with phosphate buffer was added and the cells were fixed for 15 minutes. After cell fixation, the cells were washed with phosphate buffer and treated with 0.1% Triton X-100 for 10 minutes to increase the permeability. After removing the Triton X-100 residue with phosphate buffer, the cells were blocked with 5% BSA (bovine serum albumin) diluted in phosphate buffer containing 0.2% Tween-20, and the BrdU antibody was washed with 1% BSA The cells were diluted 1: 500 and treated for one day. After removing the remaining antibody from phosphate buffer solution, the secondary antibody with fluorescence was diluted 1: 1000 with 1% BSA and treated for 1 hour. After removing the remaining antibody from the phosphate buffer solution, the mounting solution (ProLong® Gold Antifade Reagent with DAPI) containing the nuclear dye DAPI (purchased from Invitrogen) was applied to the cells. The stained cells were stored at -20 ° C, and fluorescence observation of the cells was observed at 400-fold using an inverted microscope. The results are shown in Fig.

As shown in FIG. 8, treatment with the control group BIO had no effect on cell division, whereas treatment with 20 ( R ) -ginsenoside Rh2, one of the ginsenosides derived from ginseng extract, (B) and myocardial cell (C) isolated from newborn mouse increased the number of dividing cells.

Example 11: Confirmation of expression of cyclin dependent kinase inhibitor < RTI ID = 0.0 >

The cultured C2C12 cells were plated on tissue culture plates (BD Falcon, NJ, USA) and cultured for 24 hours. Twenty-four hours later, 20 ( R ) -ginsenoside Rh2, one of the ginsenosides derived from ginseng extract, was adjusted to a concentration of 5 / / ㎖ or 10 / / ㎖, treated with BIO to 5 uM and cultured for 72 hours. After culturing for 72 hours, the cells were collected, RNA was extracted using a TRI-solution kit (BIO SCIENCE), and cDNA was synthesized using 1 μg of RNA. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on the cyclin-dependent kinase inhibitors p27, p57 and p21 using the synthesized cDNA as a template. GAPDH was used as a control group.

The DNA sequences of the primers for each gene amplification in the reverse transcription PCR are as follows.

p27 forward primer 5'-GAG TCA GCG CAG GTG GAA TTT C-3 '

p27 Reverse primer 5'- GCG AAG AAG AAT CTT CTG CAG C-3 '

p57 forward primer 5'-GCC GGT CGA GGA GCA GAA TG-3 '

p57 Reverse primer 5'-CCT GGA GGG ACG TCG TTC GA-3 '

p21 forward primer 5'- AAC ATC TCA GGG CCG AAA A-3 '

p21 Reverse primer 5'- TAA GTT TGG AGA CTG GGA GAG G-3 '

GAPDH forward primer 5'-TGA TGA CAT CAA GAA GGT GAA G-3 '

GAPDH reverse primer 5'-TCC TTG GAG GCC ATG TAG GCC AT-3 '

The reverse transcription PCR conditions are as follows.

p27 DNA Denaturation 95 캜, 15 sec; Primer Binding (Anealing) 57.8 占 폚, 15 seconds; DNA synthesis Elongation 72 캜, 30 sec; 35 cycles

p57 DNA Denaturation 95 캜, 15 sec; Anealing 58.8 캜, 15 sec; DNA synthesis Elongation 72 캜, 30 sec; 35 cycles

p21 DNA Denaturation 95 캜, 15 sec; Anealing 58.8 캜, 15 sec; DNA synthesis Elongation 72 캜, 30 sec; 35 cycles

GAPDH DNA Denaturation 95 캜, 15 sec; Anealing 58.2 占 폚, 15 seconds; DNA synthesis Elongation 72 캜, 30 sec; 35 cycles

Western blots were also performed to confirm the degree of protein expression of the cyclin-dependent kinase inhibitor. Each group of cells was extracted with a cell lytic buffer (Sigma) and 30 μg of each protein was subjected to SDS-PAGE (polyacrylamide gel electrophoresis) After separating by size, they were transferred to a membrane. The membranes were blocked with 5% skim milk powder at room temperature for 1 hour and reacted with primary antibodies corresponding to p21, p27 and p57 at room temperature for 2 hours. After washing the primary antibody and reacting with the secondary antibody, the content of each protein was confirmed by ECL (enhanced chemiluminescence system). Alpha-tubulin was used as a quantitative control.

In FIG. 9 (A), the degree of RNA expression of the cyclin-dependent kinase inhibitors p21, p27 and p57 decreases when 20 ( R ) -ginsenoside Rh2 is treated. In addition, as shown in FIG. 9 (B), the degree of protein expression of p21, p27 and p57 was decreased by 20 ( R ) -ginnenoside Rh2.

<Example 12: Toxicity test>

Example 12-1. Acute toxicity

Toxicity of the butanol fraction and 20 ( R ) -ginnenoside Rh2 compound after the ginseng enzyme treatment of the present invention to an animal body within a short period of time (within 24 hours) was investigated and the mortality rate was determined This experiment was performed. Twenty mice of the general mouse ICR mouse line were assigned to each of the control and experimental groups. In the control group, only the PEG-400 / Tween-80 / ethanol (8/1/1, v / v / v) was administered and in the experimental group, the butanol fraction after the treatment with the ginseng enzyme and the 20 ( R ) (2 g / kg / day) of 20 mg / kg / day, respectively, by oral administration. After 24 hours of administration, the mice were found to survive in the control group and in the experimental group treated with 2 g / kg / day of the ginseng enzyme-treated butanol fraction and 20 ( R ) -ginenoside Rh2 compound .

Example 12-2. Organ organs toxicity test in experimental group and control group

In the long-term toxicity test, the butanol fraction and the 20 ( R ) -ginenoside Rh2 compound of the present invention treated with the ginseng enzyme were administered to C57BL / 6J mice (10 rats per group) for 8 weeks at 2 g / kg / Respectively. ( R ) -ginsenoside Rh2 compound and PEG-400 / Tween-80 / ethanol (8/1/1) were administered to the animals. (Vital Scientific NV, Netherland) were used to determine the concentration of glutamate-pyruvate transferase (GPT) and blood urea nitrogen (BUN) in the blood after 8 weeks from the control group, Were measured using the instrument. As a result, GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to renal toxicity, showed no significant difference compared to the control group. In addition, liver and kidney were cut from each animal, followed by a general tissue section preparation, histological observation with an optical microscope, and no abnormalities were observed in all tissues.

&Lt; Formulation Example 1 >

Formulation Example 1-1. Manufacture of tablets

After the treatment of the ginseng enzyme of the present invention, 20 g of the butanol fraction or the compound 200 mg of 20 ( R ) -ginsenoside Rh2 were mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

Formulation Example 1-2. Injection preparation

100 mg of the compound 20 ( R ) -ginchenoid Rh 2 of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

<Formulation Example 2: Food Preparation>

Formulation Example 2-1. Manufacture of cooking seasonings

After the treatment with the ginseng enzyme of the present invention, the butanol fraction and the compound 20 ( R ) -ginsenoside Rh2 was added to the cooking seasoning at 1 wt%, respectively, to prepare a cooking sauce for health promotion.

Formulation Example 2-2. Manufacture of flour food products

After the treatment with the ginseng enzyme of the present invention, the butanol fraction and the compound 20 ( R ) -gincenoside Rh2 were added to flour at 0.1 wt%, respectively, and breads, cakes, cookies, crackers and noodles were prepared using this mixture to prepare foods for health promotion.

Preparation Example 2-3. Manufacture of soups and gravies

After the treatment with the ginseng enzyme of the present invention, the butanol fraction and the compound 20 ( R ) -ginsenoside Rh2 was added to the juice to 0.1 wt%, respectively, to prepare health promotion soup and juice.

Formulation Example 2-4. Manufacture of dairy products

After the treatment with the ginseng enzyme of the present invention, the butanol fraction and the compound 20 ( R ) -ginsenoside Rh2 was added to milk in an amount of 0.1 wt%, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Vegetable juice manufacturing

After the treatment with the ginseng enzyme of the present invention, the butanol fraction and the compound 20 ( R ) -ginsenoside Rh2 were added to 1,000 ml of tomato juice or carrot juice, respectively, to prepare health promotion vegetable juice.

Formulation Example 2-6. Manufacture of fruit juice

After the treatment with the ginseng enzyme of the present invention, the butanol fraction and the compound 20 ( R ) -ginsenoside Rh2 were added to 1,000 ml of apple juice or grape juice to prepare health promotion fruit juice.

Claims (14)

Characterized in that it comprises at least one ginsenoside selected from the group consisting of 20 ( R ) ( S ) -ginenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3 of the following formula (1) Or a pharmaceutically acceptable salt thereof, for the prophylaxis or treatment of sarcopenia.
[Chemical Formula 1]

The method according to claim 1,
20 ( R ) -ginsenoside Rh2. The pharmaceutical composition for preventing or treating sarcopenia diseases by promoting regeneration of muscle cells. 3. The method according to claim 1 or 2,
The pharmaceutical composition for preventing or treating muscle hypoxia diseases according to the present invention is for preventing or treating muscle hypoxia, muscle tissue disorders, cardiovascular diseases, ataxia, muscular pain, muscular dystrophy diseases caused by aging. delete delete delete delete delete delete delete The present invention relates to a method for regenerating muscle cells characterized by comprising at least one ginsenoside selected from the group consisting of 20 ( R ) ( S ) -ginenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3 of the following formula A health functional food for preventing or ameliorating a sarcopenia disease.
[Chemical Formula 1]

12. The method of claim 11,
20 ( R ) -ginsenoside Rh2 in order to promote the regeneration of muscle cells to prevent or ameliorate sarcopenia diseases. The present invention relates to a method for regenerating muscle cells characterized by comprising at least one ginsenoside selected from the group consisting of 20 ( R ) ( S ) -ginenoside Rh2, ginsenoside Rk2 and ginsenoside Rh3 of the following formula Animal drugs for the prevention or treatment of sarcopenia diseases.
[Chemical Formula 1]

14. The method of claim 13,
20 ( R ) -ginsenoside Rh2, which is an animal drug for preventing or treating sarcopenia diseases by promoting regeneration of muscle cells.

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