KR101548605B1 - Compositions comprising fractions of Panax ginseng or ginsenosides isolated therefrom for prevention or treatment of disease through activation of sirtuins - Google Patents

Abstract

The composition of the present invention comprising the fraction of Panax ginseng or a composition containing ginsenosides isolated therefrom has an effect of activating sirtuins enzyme and is associated with an increase in cell life through action on energy metabolism Cosmetics and foods for the prevention or treatment of various diseases associated with aging, diabetes, neurodegenerative diseases or mitochondria, which are diseases that are caused by the diseases such as asthma and the like.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating a ginseng fraction or a ginsenoside-containing ginsenoside-containing disease,

The present invention relates to a composition for preventing or treating diseases treated with sirtuins activation containing ginseng fractions or ginsenosides isolated therefrom.

More specifically, the ginseng extract obtained by extracting and 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, and then hexane, ethyl acetate, The present invention also relates to a composition for preventing or treating diseases treated with sirtuin activation containing a ginseng fraction obtained by adding a solvent selected from the group consisting of ethanol, ethanol and butanol, or ginsenoside isolated therefrom.

Population aging is one of the largest 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, and it is expected to grow to 155 trillion yen in 20 years (2000 "Report on the Vision of Economic and Industrial Policy in the 21st Century" . In Korea, the elderly population of 11.0% is expected to reach 24.3% by 2030 as of 2010, and urgent measures are needed.

Aging involves changes in biocomponents such as genes, proteins, and lipids. That is, aging is a phenomenon in which biochemical and biological functions of the biocomponent component are changed by interaction with various physicochemical environmental factors. In addition, aging is a complex and comprehensive life phenomenon in which various life phenomena such as occurrence of individuals, cell division, cell death and differentiation are progressed together. In contrast, aging has been reported to be associated with changes in extracellular epilepsy and cell membrane receptors (Graefes, Arch. Clin. Exp. Ophthalmol., 240 (12), 996-1002, 2002) .

On the other hand, since aging is an irreversible reaction, there is a belief that once aging is progressed, it can not be restored to a young state, but recent research has shown that aging can restore the young It is suggesting. As an example of this, when the caveolae structure in which expression is increased in aging cells is removed, cell proliferation is restored (J. Biol. Chem., 278 (30), 27789-27795, 2003) it has been reported that when an old cell is cultured in an extracellular matrix in which a young cell has been cultured, the morphology of the cell is transformed into a young state (J. Biol. Chem., 279 (40 ), 42270-42278, 2004).

One of the most important organs involved in aging is the mitochondria, which is related to the mechanism by which aging-related mechanisms regulate energy metabolism. Mitochondria regulate the expression of proteins called sirtuins (SIRTs) in order to inhibit senescence and maintain a constant level of NAD ⁺ (nicotinamide adenine dinucleotide ⁺ ). In mammalian cells, seven sirtuin proteins have been reported (Sirtuins 1 to 7), among which sirtuin 1 is present in the nucleus, and various proteins are used as substrates to induce metabolism and apoptosis, (FOXO), which regulates cell survival, glucose homeostasis (glucose homeostasis), glucose uptake (glucose uptake), and cell survival It has been reported that the activity of these proteins is regulated by removing the acetyl group from PGC-1α (peroxisome proliferating activated receptor, gamma, coactivator 1 alpha), which regulates hemeostasis, and p300, a transcriptional regulator (PLoS One. (10), e7350, 2008).

[Reference Figure 1]

In addition, as an example that activation of sirtuin can be used for the treatment of aging, administration of resveratrol (known to activate sirtuin protein) to yeast, Drosophila, C. elegans , etc. (Nature, 425 (6954), 191-196, 2003) has been reported to extend the life span by up to 59%. In addition, administration of resveratrol to mice has shown that the resveratrol-treated group has a prolonged life-span effect and significantly increased activity. In other words, mice consuming resveratrol were found to have improved muscular performance compared to the control group, as the muscles became stronger and ran twice as far. In addition, U.S. Patent No. 7977049 and U.S.P. No. 7544497 disclose that sirtuin is involved in controlling life span and stress. In addition, U.S. Patent No. 8242171 discloses that resveratrol can regulate weight by controlling sirtuin, and U.S. Patent No. 8017634 discloses that the sirtuin-regulating compound inhibits insulin resistance or metabolic diseases such as diabetes And US Patent Publication No. 2006-0229265 discloses that the sirtuin-activating compound is useful for the treatment of diseases associated with aging, stress, diabetes, obesity, degenerative brain disease, heart disease, blood coagulation, inflammation, cancer And the like can be used as a therapeutic agent.

More important than inhibiting senescence is the question of quality of life associated with the decline of geriatric disease (Cell, 127 (6), 1109-1122, 2006). Thus, And it has been reported that the quality of life can be improved by significantly reducing adult diseases related to diabetes, heart disease, fatty liver, etc., and sirtuin protein is highlighted as an important gene related to anti-aging and healthy life.

On the other hand, diseases associated with abnormal actions of mitochondria include metabolic diseases including disorders of nervous and muscular tissues and diabetes, which are also related to aging. In addition, the other mitochondrial-related diseases include at least one of neuropsychiatric diseases, visceral diseases, endocrine diseases, sensory diseases, joint diseases and muscle diseases caused by a decrease in mitochondrial activity. Such neuropsychiatric disorders include, but are not limited to, dementia, Parkinson's disease, stroke, neuropsychiatric disorders, autism, mental retardation, seizures, stroke-like seizures, migraine and neuropathic pain. The intestinal disease may include one or more of liver failure, intestinal pseudo-obstruction, irritable bowel syndrome, gastroesophageal reflux disease, constipation, diarrhea, renal tubular acidosis, and fanconi syndrome But are not limited thereto. The endocrine system disease includes, but is not limited to, one or more of pancreatic exocrine dysfunction, hypothyroidism, hypoparathyroidism, hypogonadism, developmental delay, type 2 diabetes and hypoglycemia It is not. The sensory disease includes, but is not limited to, at least one of optic atrophy, strabismus, retinitis pigmentosa, blindness, and hearing loss. The joint disease includes, but is not limited to, one or more of degenerative arthritis, rheumatoid arthritis, and osteoarthritis. The muscle disorder may be ataxia, dysarthria, dysphagia, spasticity, cerebral mastication, muscular pain, muscular dystrophy, muscle spasm, ptosis, ocular muscle paralysis, progressive ophthalmic paralysis, Atopic dermatitis, Leber's hereditary optic neuropathy, respiratory disorders, impaired reflexes, and autonomic ataxia.

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). This medicine has a peculiar odor, taste is slightly sweet, 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 to increase the anxiety and renal function.

On the other hand, the inventors of the present invention have found that ginsenoside fractions or ginsenosides derived from the fractions activate various kinds of diseases associated with abnormal actions of aging, diabetes, neurodegenerative diseases or mitochondria by activating the sirtuin enzyme, The present invention has been accomplished by confirming that there is an effect of preventing or treating a related disease.

Prior art patents related to sirtuin activity include "a composition containing a mulberry extract (Korean Patent No. 826209)", "thiazolopyridine-sirtuin-regulating compound" (Korean Patent Publication No. 2011-0110194) (Korean Patent Publication No. 2011-0102468), "isoindolinone and related analogues as a sirtuin modulator (Korean Patent Laid-Open No. 2011-0098789) "," Quinazolinone, quinolone and related analogues (Korean Patent Publication No. 2011-0065536) as a sirtuin regulator, "" Pyridine, bicyclic pyridine and related analogues (Korean Patent Publication No. 2011-0079763) (Korean Patent Laid-Open No. 2011-0063573) as a sirtuin modulator (Korean Patent Publication No. 2011-0063573), benzoxazole, benzthiazole and related analogs as a sirtuin modulating agent (Korean Patent Publication No. 2011-0044291) , The compounds disclosed in these patents, And its structure and properties are different from those of the ginsenoside compound.

In addition, it is known that ginseng extract (Korean Patent Laid-Open No. 2011-0131783) processed at high temperature and high pressure containing various ginsenosides has an anti-aging effect on the skin, but the extracting conditions are different from those of the ginseng fraction of the present invention, The effect of the single ginsenoside as in the present invention is not disclosed. In addition, the skin aging inhibiting effect, which inhibits keratinocyte damage described in the above-mentioned prior arts, and the lifetime extension and aging suppression effects of activation of the syrup such as the present invention are completely different.

Furthermore, although ginsenoside Rg ₃ has been shown to inhibit apoptosis of endothelial cells through the caspase pathway of mitochondria, it has been shown that vascular disease associated with vascular endothelial cells It is also possible to say that the invention is completely different from the invention in terms of technology.

Korean Registered Patent No. 826209 (composition containing an acid mulberry extract, registered on Apr. 23, 2008) Korean Patent Laid-Open No. 2011-0110194 (Thiazopyridine-Syrupine Modulating Compound, published on Jun. 10, 2011) Korean Patent Publication No. 2011-0102468 (Phthalazinone and Related Analogues as a Sirtuin Regulator, published on Sep. 16, 2011) Korean Patent Publication No. 2011-0098789 (isoindolinone and related analogs as a sirtuin modulator, published on Sep. 1, 2011) Korean Patent Publication No. 2011-0079763 (pyridine, bicyclic pyridine and related analogues as a sirtuin modulator, published July 7, 2011) Korean Patent Publication No. 2011-0065536 (quinazolinone, quinolone and related analogs as a sirtuin modulator, published on June 15, 2011) Korean Patent Publication No. 2011-0063573 (chromenone analog as a sirtuin modulator, published Jun. 10, 2011) Korean Patent Publication No. 2011-0044291 (benzoxazole, benzthiazole and related analogues as a sirtuin modulator, published on April 28, 2011) Korea Patent Publication No. 2011-0131783 (cosmetic composition for preventing aging of skin containing processed ginseng extract as an active ingredient, disclosed in Dec. 07, 2011) U.S. Patent No. 8242171 (Method for reducing the weight of a subject or inhibiting weight gain in a subject. U.S. Patent No. 8017634 (Registered on September 13, 2011 for treating obesity and insulin resistance disorders) U.S. Patent No. 7977049 (Registered on July 12, 2011) (Methods and compositions for extending the life span and increasing stress resistance of cells and organisms) U.S. Patent No. 7544497 (registered with Jun. 9, 2009 for Compositions for manipulating the lifespan and stress response of cells and organisms) US Patent Publication 2006-0229265 (Nicotinamide riboside and analogues thereof.

Cho, K.A. et al., Senescent phenotype can be reversed by reduction of caveolin status., J. Biol. Chem., 278 (30), 27789-27795, 2003. Cho, K.A. et al., Morphological adjustment of senescent cells by modulating caveolin-1 status., J. Biol. Chem., 279 (40), 42270-42278, 2004. Human sirt-1: molecular modeling and structure-function relationships of an unordered protein., PLoS One., 4 (10), e7350, 2008. Autiero I, et al. Howitz, K.T. et al., Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan., Nature, 425 (6954), 191-196, 2003. Lagouge, M. et al., Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha., Cell, 127 (6), 1109-1122, 2006. Kim, E.J. et al., Active regulator of SIRT1 cooperates with SIRT1 and facilitates suppression of p53 activity., Molecular Cell, 28 (2), 277-290, 2007. Min, J.K. et al., 20 (S) -Ginsenoside Rg3 prevents endothelial cell apoptosis via inhibition of a mitochondrial caspase pathway. Biochem. Biophys. Res. Commun., 2006, 349 (3), 987-994.

It is an object of the present invention to provide a composition for the prevention or treatment of diseases which are treated with sirtuin activation containing ginsenoside fractions or ginsenosides isolated therefrom.

More specifically, the object of the present invention is to provide a ginseng extract obtained by suspending ginseng in water, extracting a ginseng extract with water, a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof, There is provided a composition for the prevention or treatment of diseases treated with sirtuin activation comprising a ginseng fraction obtained by adding a solvent selected from the group consisting of hexane, ethyl acetate, and butanol or ginsenoside isolated therefrom have.

The present invention relates to a pharmaceutical composition comprising Panaxol A (Compound 1 ), Panaxol B (Compound 2 ), Panaxol C (Compound 3 ), Dammar-20 (22) diene -3 β, 6 α, 12 β - triol (Dammar-20 (22), 24-diene-3 β, 6 α, 12 β -triol, compound 4), and, 20 (S) - ginsenoside Rg ₃ (20 (S ) -Ginenoside Rg ₃ , ( 5 ), which comprises a ginseng fraction containing at least one ginsenoside selected from the group consisting of the compound ( 5 ).

[Chemical Formula 1]

The ginseng fraction 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 adding to the suspension a mixture of hexane, ethyl acetate, And an organic solvent selected from the group consisting of ethanol, ethanol, and butanol. More preferably, the ginseng fraction is obtained 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, A concentrate of a hexane layer, a concentrate of an ethyl acetate layer obtained by mixing ethyl acetate with a residue obtained by removing the hexane layer or a concentrate of a butanol layer obtained by mixing butanol with a residue obtained by removing the ethyl acetate layer.

The disease to be treated by the above-mentioned sirtuin activation may be selected from the group consisting of aging, muscle tissue disorders, metabolic diseases including diabetes, stroke, stroke-like seizure, dementia, Parkinson's disease, neurological disorders, neuropsychiatric disorders, Diabetic retinopathy, constipation, diarrhea, renal tubular acidosis, Panconi's syndrome, pancreatic exocrine dysfunction, hypothyroidism, hyperparathyroidism, hyperparathyroidism, Hypotonia, hypogonadism, hypogonadism, hypotonia, optic atrophy, strabismus, retinitis pigmentosa, blindness, hearing loss, degenerative arthritis, rheumatoid arthritis, osteoarthritis, ataxia, dysphagia, dysphagia, stiffness, , Muscular dystrophy, muscle spasms, ptosis, eye muscle paralysis, progressive oculomotor paralysis, lever optic atrophy and respiratory disorders There.

The present invention also relates to a pharmaceutical composition comprising Panaxol A (Compound 1 ), Panaxol B (Compound 2 ), Panaxol C (Compound 3 ), Dammar 20 (22), 24-diene -3 β, 6 α, 12 β - triol (Dammar-20 (22), 24-diene-3 β, 6 α, 12 β -triol, compound 4), and, 20 (S) - ginsenoside _{Rg 3 (20 (S) -Ginsenoside} Rg 3, Compound 5 ). ≪ Desc / Clms Page number 2 > The present invention relates to a composition for preventing or treating a disease treated with sirtuin activation, which comprises at least one ginsenoside selected from the group consisting of

In another aspect, the present invention provides a pharmaceutical composition comprising Panaxol A (Compound 1 ), Panaxol B (Compound 2 ), Panaxol C (Compound 3 ), Dammar-20 22), 24-diene -3 β, 6 α, 12 β - triol (Dammar-20 (22), 24-diene-3 β, 6 α, 12 β -triol, compound 4), and, 20 (S ) - ginsenoside _{Rg 3 (20 (S) -Ginsenoside} Rg 3, ( 5 ), wherein the ginseng fraction comprises at least one ginsenoside selected from the group consisting of the compound ( 5 ).

The present invention provides a novel compound having the following chemical structure separated from a ginseng fraction and comprising Panaxol A (Compound 1 ), Panaxol B (Compound 2 ) and Panaxol C (Compound 3 ) do.

It is also possible to use at least one ginsenoside selected from the group consisting of Panaxol A (Compound 1 ), Panaxol B (Compound 2 ), and Panaxol C (Compound 3 ) A method of separating the side can also be provided.

Preferably, the ginseng extract obtained by extracting and 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, and then hexane, ethyl acetate, (Panaxol A, Compound 1 ), paraxol B (Panaxol B, compound 2 ), and paraxol C ( 1 ) were obtained from a concentrate of an organic solvent layer obtained by adding an organic solvent selected from the group consisting of Panaxol C, Compound 3 ) can be isolated from the reaction mixture.

More preferably, the ginseng extract is concentrated by extracting with water, a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof, suspending the ginseng extract in water, mixing with hexane, and concentrating the hexane layer The concentrate of the ethyl acetate layer obtained by mixing the residue obtained by removing the hexane layer with ethyl acetate or the concentrate of the butanol layer obtained by mixing the residue obtained by removing the ethyl acetate layer from the concentrate and the butanol layer (Panaxol A, One or more ginsenosides selected from the group consisting of Compound 1 ), paraxol B (Panaxol B, compound 2 ) and paraxol C (compound 3 ) can be isolated.

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 a method of isolating a number of compounds having the structure confirmed and determining whether the ginseng fraction and the compound represented by the formula 1 have a sirtuin activating action.

The process by which the ginseng fraction of the present invention is selected as a sirtuin-activating composition is as follows. Various natural plants and herbal medicines were collected and cloned into a cell to introduce a sirtuin gene into a cell to examine the activity of sirtuin, or to examine the ratio of NAD ⁺ / NADH in a cell to select ginseng ( Panax ginseng ) Ginseng extract and ginseng fraction, from which the sirtuin-activating compound is isolated.

The ginseng fraction of the present invention can be prepared by first preparing a concentrated ginseng extract, preparing a suspension by adding water to the concentrated ginseng extract, and then fractionating it with various solvents to prepare a fraction. The extraction solvent for the preparation of the extract is preferably 1 to 30 times the weight of ginseng. In addition, when the concentrated ginseng extract is 0.0001 to 10 g, it may be mixed with 1 to 10 L of water to prepare a suspension, and then various fractions can be prepared by adding various solvents. 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 by weight based on the weight of the suspended ginseng extract.

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 purifying and purifying a compound having a sirtuin activating effect 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 is involved in the activation of sirtuin and thus is associated with diseases associated with an increase in cell life, that is, an abnormal function of aging, diabetes, neurodegenerative diseases and mitochondria May be used as a composition for preventing or treating various diseases, and preferably used as a composition for preventing or treating diabetes or aging. In particular, the diabetes may be senile diabetes.

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 the prevention or treatment of diseases treated with sirtuin activation comprising ginseng fractions or ginsenosides 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 the suppository base 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 will depend on the age, sex, and weight of the subject to be treated, the severity of the particular disease or condition being treated, the severity of the disease or condition, It will depend on the judgment of the prescriber. 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 provides a health functional food for preventing or ameliorating a disease treated with sirtuin activation comprising a ginseng fraction or a food-acceptable food-aid additive containing ginsenosides isolated therefrom .

The composition of the present invention comprising the fraction of Panax ginseng or a composition containing ginsenosides isolated therefrom has an effect of activating sirtuins enzyme and is associated with an increase in cell life through action on energy metabolism Cosmetics and foods for the prevention or treatment of various diseases associated with aging, diabetes, neurodegenerative diseases or mitochondria, which are diseases that are caused by the diseases such as asthma and the like.

C] 및 (B) NOESY [H

H]의 상관관계를 나타내는 그림이다.
도 2는 본 발명의 화합물 1~5의 시르투인1 활성을 루미노센스(luminescence)를 이용하여 확인한 결과를 나타내는 그림이다.
도 3은 HEK293 세포에서 루시퍼라아제 리포터 어세이를 이용하여 본 발명의 화합물 1~5의 시르투인1 활성이 p53에 미치는 영향을 확인한 결과를 나타내는 그림이다.
도 4는 HEK293 세포에서 NAD⁺/NADH 비율을 통해 본 발명의 화합물 1~5의 시르투인1의 활성을 확인한 결과를 나타내는 그림이다.
도 5는 β-갈락토시다아제에 의하여 염색된 섬유아세포를 찍은 사진이다. Figure 1 (A), HMBC [H of the compounds of the invention 1-3 (novel compound)

C] and (B) NOESY [H

H].
2 is a graph showing the results of confirming the activity of the compounds of the present invention 1 to 5 by using luminescence.
3 is a graph showing the results of confirming the effect of the compounds 1 to 5 of the present invention on the p53 activity using the luciferase inhibitor assay in HEK293 cells.
FIG. 4 is a graph showing the results of confirming the activity of Sirtuin 1 of Compounds 1 to 5 of the present invention through the ratio of NAD ⁺ / NADH in HEK293 cells. FIG.
Fig. 5 is a photograph of a fibroblast stained with? -Galactosidase.

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 and fractions >

The ginseng used in the present invention was dried in the local market of Gwangju. 100 g of the dried ginseng was extracted with 1000 ml of a solvent such as distilled water, ethanol, methanol, acetone and the like using a sonicator for 3 hours for 4 hours. Thereafter, the extracts from which the solid content was removed were concentrated under reduced pressure to obtain respective extracts. Separation conditions of the ginseng fraction and ginsenoside of the present invention were established through the activity confirmation method of the sirtuin 1 disclosed in Example 4. That is, after confirming the effect of the sirtuin 1 activity of the ginseng extract shown in the following Table 1, the activity of the 90% ethanol aqueous solution and the 70% methanol aqueous solution were further fractionated to compare the activity of the sirtuin 1. To this end, the ginseng extracts were each suspended in water (2 L) and mixed with hexane (2 L). Concentration of a hexane layer obtained by removing the hexane layer, and concentration of ethyl acetate layer obtained by mixing ethyl acetate (2 L) A concentrate of the butanol layer obtained by mixing water and the residue obtained by removing the ethyl acetate layer (2 L) was obtained, and the concentrates were used as fractions under the respective conditions. Furthermore, as shown in Table 2, the activity of the Sirtuin 1 activity of the fractions obtained by sequentially fractionating 90% aqueous ethanol solution and 100% methanol extract with hexane, ethyl acetate and butanol was confirmed. As a result, It can be confirmed that the activity of Toluin 1 is good, and especially, the effect is best in the butanol fraction.

extract Extraction quantity
(Extraction amount g per 100 g of dry sample) Active measurement concentration
(占 퐂 / ml) Non-treatment group preparation
Effect of sirtuin 1 activity (%) 30% ethanol aqueous solution extract 4.02 g 20 125 50% ethanol aqueous solution extract 4.40 g 20 130 70% ethanol aqueous solution extract 3.89 g 20 122 90% ethanol aqueous solution extract 4.01 g 20 134 100% ethanol extract 3.13 g 20 130 100% methanol extract 3.06 g 20 126 100% acetone extract 0.86g 20 119 Distilled water extract 4.36 g 20 101 Untreated group (control group) - - 100

Condition Active measurement concentration
(占 퐂 / ml) (%) Activity of sirtuin 1 relative to untreated group 90% ethanol aqueous solution extract 20 134 Of 90% aqueous ethanol solution
Hexane fraction 20 89 Of 90% aqueous ethanol solution
Ethyl acetate fraction 20 103 Of 90% aqueous ethanol solution
Butanol fraction 20 153 Untreated group (control group) - 100

< Example  2. Ginseng Butanol Fraction  Manufacturing and Ginenoside  Separation>

The ginseng extract was obtained by immersing dried ginseng (206 g) in a 90% ethanol aqueous solution at room temperature for one week at room temperature. The extract was concentrated under reduced pressure to obtain ginseng extract (19.6 g). The ginseng extract was suspended in 2 L of water, mixed with hexane (2 L), concentrated under reduced pressure, and the ethyl acetate layer obtained by mixing the residue (water layer) from which the hexane layer was removed with ethyl acetate (2 L) A pressure-reduced concentrate of the butanol layer obtained by mixing the reduced pressure concentrate and the residue (water layer) from which the ethyl acetate layer had been removed was mixed with butanol (2 L), and the reduced pressure concentrates were used as fractions under the respective conditions. Among them, ginsenosides were separated from the ginseng butanol fraction (7.2 g). After isolating and purifying the active substance, the butanol fraction (6.5 g) was subjected to silica gel column chromatography (3 × 50 cm; 63-200 mm particle size) of EtOAc / MeOH (10: 0 → 0: 1) And divided into seven small fractions (F1-F7). The small fraction F1 (503 mg) was purified by RP-18 column chromatography (1 x 30 cm; 40-63 mm particle size) of MeOH / H ₂ O (1/3 → 1/0) (F1.1-F1.4). The small fraction F1.3 (103 mg) was subjected to RP-18 column chromatography (1 x 30 cm; 40-63 mm particle size) of MeOH / H ₂ O (1/2 → 1/1) (F1.4.1-F1.4.6). Small fraction F1.4.2 (23 mg) was analyzed by HPLC (Optima Pak C ₁₈ column (10 × 250 mm, particle size 10 μm, RS Tech, Korea); _{mobile phase MeOH in H 2 O containing} 0.1% HCO 2 H (0-40 min: 62% MeOH); flow rate 2 ml / min; UV detection at 205 and 254nm] the embodiment to give the compound _{2 (t R = 32min, 1.9㎎} ) and compound _{3 (t R = 36min, 2.3㎎} ).

The small fraction F1.4.5 (20 mg) was purified by RP-18 column chromatography (1 x 30 cm; 40-63 mm particle size) using solvent conditions of MeOH / H ₂ O Compound 1 (2.0 mg) was obtained. Small fraction F1.4.6 (19㎎) to _{MeOH / H 2 O (1/2 →} 1/0) of the RP-18 column chromatography using a (1 × 30㎝ 40-63㎜ particle size) of the compound 4 ( 3.1 mg). The small fraction F3 (701 mg) was subjected to silica gel column chromatography (1 × 30 cm; 63-200 mm particle size) of CHCl ₃ / MeOH (40: 0 → 0: 1) and divided into seven small fractions (F3 .1-F3.7).

Compound _{5 (t R = 40min, 3.3㎎} ) is a small fraction F3.4.8 (26㎎) HPLC [Optima Pak C 18 column (10 × 50 mm, particle size 10 μm, RS Tech, Korea); _{mobile phase MeOH in H 2 O containing} 0.1% HCO 2 H (0-40min: 80% MeOH); flow rate 2 ml / min; UV detection at 205 and 254 nm].

&Lt; Example 3: Physicochemical properties of ginsenoside derived from ginseng >

The structure of the ginsenosides isolated in Example 2 was analyzed by molecular weight obtained by using an ESI mass spectrometer (ESI mass spectrometer) and the results of ¹ H-NMR and ¹³ C-NMR analysis of nuclear magnetic resonance Respectively. The structures of the compounds of novel compounds 1-3 in the ginsenoside is a further nuclear magnetic resonance ^{(NMR) 1 H-NMR,} 13 C-NMR, HMQC (1 H-detected heteronuclear multiple-quantum coherence) through analysis, HMBC ( heteronuclear multiple-bond coherence, and distortionless enhancement by polarization (DEPT) spectra and determined the molecular structure.

Example 3-1. Paradoxal A (compound 1)

Panaxola;

Brown, amorphous powder;

+25.01 (c 0.3, MeOH); [alpha]

+25.01 (c 0.3, MeOH);

IR (KBr)? _Max 3397, 1722, 1705, 1654, 1381, 1182, 982, 905 cm ^-1 ;

¹ H NMR data (500 MHz) and ¹³ C NMR data (125 MHz) - see Table 3 ;

HRFABMS m / z 495.3457 (calcd for C 30 H 48 O 4 Na, 495.3450).

Compound 1 was isolated as a brown powder, the molecular formula was determined from the ^{[M + Na] + (calcd} for C 30 H 48 O 4 Na, 495.3450) from the measured value of HRFABMS as C ₃₀ H ₄₈ O ₄ 495.3457. Compound 1 was able to confirm strong IR absorbance at ν _max OH (3397 cm ^-1 ), C═O (1722, 1705 cm ^-1 ) and C═C (1654 cm ^-1 ). ¹ H NMR analysis of Compound 1 revealed that 6 methyl groups were present at δ _H 1.40, 1.39, 1.30, 1.17, 0.90 and 0.89 and methyl groups δ _H 4.15 (td, J = 10.5, 3.5) bonded to olefinic at δ _H 1.73 and 1.67 Hz), and one olefinic proton at δ _H 5.15 (t, J = 6.5 Hz). ^{The 13} C-NMR results showed two ketone carbonyl carbons at δ _C 218.4 and 212.5, two olefinic carbons at δ _C 131.7 and 124.6, one oxymethine carbon at δ _C 67.6 ) And 30 carbons including quaternary carbon to which sp ³ type oxygen is connected at? _C 73.3.

Detailed ¹ H and ¹³ C NMR analysis of the compound 1 suggests that the compound 1 is wave incident dione (panaxadione) similar to the A / B ring structure of the compound. In addition, C / D shape of a ring and a substituent of Compound 1 3- O of four triol (betulafolenetriol) in betu rapport-confirmed that similar to the keto-acetyl -12- (3- O -acetyl-12-keto ) derivative Respectively. The predicted structure above shows that the two carbonyl groups are attached to carbon C-3 and C-12 respectively, the two hydroxyl groups are bonded to C-6 and C-20, and the olefin groups to C-24 and C-25 . HMBC experiments were performed to confirm the exact structure of the substituents. Hydrogen _{H-2 (δ H 2.78)} , H 3 -28 (δ H 1.39), H 3 -29 (δ H 1.40) from a carbon position _C-3 (δ C 218.4) connected to a, _H-5 (δ H 1.78) and _H-7 (δ H 1.92) from the _{C-6 (δ C 67.6)} , H-11 (δ H 2.29) and _C-12 (δ C 212.5) from the _{H-13 (δ H 2.95)} , H- 17 (δ _H 2.47) and _{H 3 -21 (δ H 1.17)} C-20 (δ C 73.3), and _{H-24 (δ H 5.15)} C-26 (δ C 25.7) and C-27 (δ from from _C 17.1) confirms that the above estimates are correct.

Hydrogen H-5 and H-6 (J = 10.5 Hz) pair values written by a constant (coupling constant) of the H-6 the H-5 and axes forming (axial) was estimated that the relationship, additional H-6 from the H ₃ - 19 and H ₃ -29, we determined that the hydroxyl groups at position 6 were arranged at the α - position. Hydrogen H-13 and H-17 (J = 10.0 Hz ) , and NOESY connection to the H ₃ -21 H-17 from the H-17 is α - it was confirmed that the three-dimensional array arranged in a C-20 (S). From the above results, Compound 1 is determined as a novel compound having 3- O -acetyl-12-keto derivatives of panaxadione and betulafolenetriol. Thus, the Compound 1 6 α, 20 (S) - dihydroxy wall Mar -3,12- dione -24- yen (6 α, 20 (S) -dihydroxydammar-3,12-dione-24-ene) (Panaxol A), which has a structure as shown in Fig.

Example 3-2. Parapsule B (Compound 2)

Panaxol B;

Brown, amorphous powder;

-51.5 (c 0.2, MeOH); [alpha]

-51.5 (c 0.2, MeOH);

IR (KBr)? _Max 3362, 2971, 1721, 1700, 1637, 1586, 1421, 1181, 1023, 905, 670 cm ^-1 ;

¹ H NMR data (500 MHz) and ¹³ C NMR data (125 MHz) - see Table 3 ;

HRFABMS m / z 511.3409 (calcd for C ₃₀ H ₄₈ O ₅ Na, 511.3399).

Compound 2 was determined from was isolated as a brown powder, its molecular formula is C ₃₀ H ₄₈ O ₅ to the [M + Na] ⁺ of at measure of _{511.3409 HRFABMS (calcd for C 30 H} 48 O 5 Na, 511.3399) . Compound 2 was able to confirm strong IR absorbance at ν _max OH (3362 cm ^-1 ), C═O (1721, 1700 cm ^-1 ) and C═C (1637 cm ^-1 ). ¹ H and ¹³ C NMR analysis of the compound 2 was confirmed that a side which is a substituent hydroxy group in C-3 and C-12 single two ketone groups and C-6 of the compound [1] is connected similarly. This result is, H ₂ -2 HMBC of the compound 2 (δ _H 2.79 and _{2.31), H 3 -28 (δ} H 1.67), H 3 -29 C-3 (δ C 218.8) from (δ _H 1.71) , and _{H-11 (δ H 2.29)} , H-13 (δ H 3.41), H-17 (δ H 2.80) from the _{C-12 (δ C 211.9)} , H-5 (δ H 1.94) and H-7 (delta _H 1.87) to C-6 (delta _C 67.2).

The ¹ H and &lt; ¹³ &gt; C NMR analyzes of Compound 2 additionally show the presence of two hydroxyl groups and one olefin group from the molecular formula in addition to the two ketone groups and one hydroxyl group. Deshielding of methyl groups observed at δ _C 110.5 carbons and δ _H 1.90 (brs) due to olefin hydrogen in a pair of δ _H 5.28 (brs) and 4.97 (brs) identified from the ¹³ C NMR analysis results ) ^{Suggests that} the presence of a Δ ^25,26 olefin system and that the methyl group observed at δ _H 1.90 is hydrogen due to H ₃ -27. These results suggest that _{H 3} -27 (δ H 1.90) from the _C-25 (δ C 150.7) and _{C-26 (δ C 110.5)} HMBC connection to, _{H 2} -26 (δ H 5.28 and 4.97), H ₃ - HMBC coupling from 27 (delta _H 1.90) to delta _C 76.2 confirmed that the position of the carbon at delta _C 76.2 was C-24 and that hydroxyl groups were present. Position of the additional hydroxyl group is that the _H-13 (δ H 3.41) in the No. _{20, H-17 (δ H 2.80} ), H 3 -21 HMBC of the _C-20 (δ C 73.7) from (δ _H 1.46) Lt; / RTI &gt;

The stereochemistry of compound 2 was confirmed by the coupling constant, NMR analysis of existing literature values, and NOESY experiments. Mating constant of the H-5 and H-6 (J = 10.1 Hz) H-6 are, and the axial relationship between H-5 and 6 have a single hydroxyl group-H- that the arrangement - and provided that the arrangement, H-17 is also a 13 and H-17 ( J = 10.0 Hz). The NMR results of C-17 ring, C-20 ring, C-21 ring and C-22 ring of compound 2 and 3- O -acetyl-12- ( S ), and these results were confirmed from the NOESY linkage from H-6 to H ₃ -19 and H ₃ -29, H-17 to H ₃ -21. Thus, Compound 2 is 3,12- dione -6α, 20 (S), 24- trihydroxy wall Mar -25- yen (3,12-dione-6α, 20 (S), 24-trihydroxydammar-25-ene ) Was named Panaxol B (Panaxol B).

Example 3-3. Parapsol C (Compound 3)

Panaxol C;

Brown, amorphous powder;

+25.01 (c 0.3, MeOH); [alpha]

+25.01 (c 0.3, MeOH);

IR (KBr)? _Max 3361, 2972, 1715, 1700, 1541, 1459, 1340, 1227, 1030, 926, 653 cm ^-1 ;

¹ H NMR data (500 MHz) and ¹³ C NMR data (125 MHz) - see Table 3 ;

HRFABMS m / z 511.3409 (calcd for C ₃₀ H ₄₈ O ₅ Na, 511.3399).

Compound 3 was determined from was isolated as a brown powder, its molecular formula is C ₃₀ H ₄₈ O ₅ to the [M + Na] ⁺ of at measure of _{511.3346 HRFABMS (calcd for C 30 H} 48 O 5 Na, 511.3399) . Compound 3 was able to confirm strong IR absorbance at ν _max OH (3361 cm ^-1 ) and C═O (1715, 1700 cm ^-1 ). Compound 3 ¹³ C NMR analysis results δ _C 46.0 _(C-22) in a chemical shift value of the chemical shift of the compound 3 Compound 2, 123.2 (C-23) , 143.3 (C-24), 70.2 (C- 25), 31.3 (C-26), and 31.2 (C-27). ¹ H NMR spectrum of compound 3 is a pair of oleic pinik results that can be estimated by proton is δ _H 6.25 (m) and 6.02 (d, J = 15.4 Hz ) oleic Δ ^25,26 of the chemical shift value of compound 2 pinik was born newly displayed in place of the proton chemical shift values of δ _H 5.28 (brs) and 4.97 (brs), also oxy methine (oxymethine) protons of _{δ H-24 4.41 (t,} J = 5.0 Hz) the compound 3, Sarah lost.

These results are from the _{H 3 -26/27 (δ H 1.53,} 6H) HMBC results of compound _{3 C-24 (δ C 143.3} ) and _{C-25 (δ C 70.2)} , H 2 -22 (δ H 2.61 and 2.54) to C-23 (? _C 123.2) and C-24 (? _C 143.3). The large binding constant of J = 15.4 Hz for Compound 3 suggests that H-23 and H-24 have an E- type of Δ ^23,24 olefin bond. Hydrogen H-5 and H-6 (J = 11.0 Hz ), H-13 and H-17 (J = 10.0 Hz ) large coupling constant and also -19 H ₃ and H ₃ from the H-6 obtained from NOESY spectra - 29, and the connection from H-17 to H ₃ -21 determines the cubic arrangement of C-6, C-17 and C-20. Thus, compound 3 is 3,12- dione -6α, 20 (S), 25- trihydroxy wall Mar -23- yen (3,12-dione-6α, 20 (S), 25-trihydroxydammar-23-ene ) Was named Panaxol C (Panaxol C).

Example 3-4. 20 (22), 24-diene-3 beta , 6 alpha , 12 beta Triol (Compound 4)

Dammar-20 (22), 24-diene-3 [ beta] , 6 [alpha] , 12 [ beta] -triol;

FAB-MS: [M + Na] &lt; ^{+ &} gt ; m / z 481.38;

^{1 H NMR data (400 MHz,} in C 5 D 5 N): 5.39 (1H, brt, J = 7.1 Hz, H-22), 5.19 (1H, brt, J = 7.1 Hz, H-24), 4.33 ( 1H, m, H-12) , 3.86 (1H, m, H-6), 3.44 (1H, dd, J = 11.3, 5.0 Hz, H-3), 2.68 (2H, dd, J = 7.5, 7.1 Hz H-23), 1.92 (3H, s, H-29), 1.73 (3H, s, H-21), 1.52 1.36 (3H, s, H-30), 1.06 (3H, s, H-28), 0.92 (3H, s, H-18), 0.87 (3H, s, H-19);

^{13 C NMR data (150 MHz,} in C 5 D 5 N): 39.6 (C-1), 28.2 (C-2), 78.4 (C-3), 40.4 (C-4), 61.8 (C-5) , 67.7 (C-6), 47.7 (C-7), 41.5 (C-8), 50.5 (C-9), 40.4 (C-13), 50.9 (C-14), 32.3 (C-15), 28.9 (C-16), 50.4 (C-20), 13.2 (C-21), 123.2 (C-22), 27.5 (C-23), 123.5 C-27), 32.0 (C-28), 16.5 (C-29), 17.7 (C-30).

Examples 3-5. 20 ( S ) - Ginsenoside Rg ₃ (Compound 5)

20 ( S ) -Ginenoside Rg ₃ ;

ESI-MS: [M + H] &lt; ⁺ &gt; m / z 785.1;

^{1 H NMR data (400 MHz,} in DMSO- d 6): 5.09 (1H, t, J = 6.5 Hz, H-24), 3.37 (1H, m, H-12), 3.01 (1H, dd, J = (1H, m, H-23), 1.87 (1H, m, H-23) (1H, m, H-16), 1.67 (1H, m, H-11) 1H, m, H-2b), 1.55 (1H, m, H-1a), 1.54 (3H, s, H- H-15a), 1.39 (1H, m, H-22a), 1.36 (1H, m, H-6b), 1.34 m, H-22b), 1.21 (1H, m, H-16b), 1.19 ), 0.97 (3H, s, H-28), 0.94 (1H, m, H-1b) s, H-30), 0.80 (3H, s, H-19), 0.73 (3H, s, H-29), 0.69 (1H, overlap, H-5); sugar part: 4.26 (1H, d , J = 5.5 Hz, H-1 '), 3.31 (1H, t, J = 5.5 Hz, H-2'), 3.34 (1H, t, J = 5.5 Hz, H- M, H-6 '), 3.07 (1H, overlap, H-4'), 3.07 'b), 4.42 (1H, d, J = 5.5 Hz, H-1''), 2.98 (1H, t, J = 5.5 Hz, H-2''), 3.12 (1H, t, J = 5.5 Hz , H-3 ''), 3.11 (1H, t, J = 5.5 Hz, H-4 ''), 3.02 ), 3.47 (1H, m, H-6 &apos;b);

^{13 C NMR data (100MHz, in} DMSO- d 6): 38.5 (C-1), 25.7 (C-2), 88.1 (C-3), 38.6 (C-4), 55.4 (C-5), 17.6 (C-6), 34.3 (C-7), 36.2 (C-8), 49.3 (C-9), 38.8 C-13), 50.4 (C-14), 30.5 (C-15), 35.8 (C-16), 53.4 C-24), 129.8 (C-25), 25.4 (C-26), 17.5 (C-22) (C-1), 81.2 (C-2), 76.4 (C-3 '), 27.4 (C-28), 15.7 , 69.9 (C-4 '), 76.3 (C-5'), 60.9 (C-6 '), 103.8 ), 69.8 (C-4 ''), 76.7 (C-5 ''), 60.7 (C-6 '').

Example 4: Measurement of sirtuin 1 enzyme activity in a test tube [

Sirtuin 1 is a protein that diacetylates the K382 portion of p53. This diacetylation activates the p53-related transcription process and ultimately counteracts apoptosis due to cell senescence and DNA damage and stress . Therefore, the degree of activation of the sirtuin 1 enzyme of the compounds 1 to 5 of the present invention was measured directly using the sirtuin enzyme.

First, the deacetylase activity of sirtuin 1 enzyme was measured using luminescence (the increase in activity of sirtuin 1 deacetylase activity and the increase in activity of sirtuin 1 have the same meaning ). To this end, Flour de-Lys-SIRT1 substrate (KI-177), Flour de-Lys-Developer II (KI-176) and SIRT1, human, recombinant, SE- Respectively. The compounds 1 to 5 of the present invention were treated with 10 to 20 占 퐂 / ml of sirtuin 1 reaction solution (500 mM Tris / Cl pH 8.0, 100 mM MgCl ₂ , 1.37 M NaCl, 270 mM KCl, 100 mg / , Siru-in 1 enzyme was added and reacted. Then, 2X substrate mixture of biomolecule was added to the reaction solution. After completion of the reaction, the sample is centrifuged and reacted with a developer II mixture. Thereafter, the activity of the protein was measured at 360/450 nm after addition of SIRT1 assay buffer, and the results are shown in FIG. At this time, the control group was treated with the same volume (within 0.1% of the cell culture medium) of DMSO, which is a solvent of the compound.

When resveratrol (molecular weight 228) of 2.3 μg / ml (10 μM) was used, the activity of persulphin was activated about 2 times and the concentration of 1.2 μg / (10 μM) nicotinamide inhibited 30-50% of the protein activity of sirtuin 1, indicating that a system capable of simultaneously measuring the active agent and the inhibitor was established.

On the other hand, it was confirmed that the compounds 1 to 5 of the present invention increase the activity of the sirtuin 1 enzyme in a concentration-dependent manner. In particular, it is confirmed that the compound 4 has a remarkably excellent sirtuin-activating function.

Example 5. Identification of the activity of sirtuin 1 using a p53 luciferase reporter assay.

When the activity of sirtuin 1 in the cell increases, the activity of p53 is decreased in the cell (Molecular Cell, 28, 277-290, 2007). That is, a method of predicting the activity of sirtuin 1 was used by measuring the activity of p53 using luciferase transfection.

HEK293 cells were incubated at 70% saturation and washed with PBS. After 0.25% of trypsin was treated at room temperature for 1 minute, cells were collected by adding DMEM and centrifuged at 2,500 rpm for 5 minutes to remove the supernatant to obtain cell precipitate. The cells were then mixed well with DMEM medium. Next, 3 μl of lipopectamine (life Technologies, Inc.) was added to 100 μl of serum-free media (DMEM), and the mixture was slowly mixed and allowed to stand at room temperature for 15 minutes. Then, 3.0 μg of p53-Luc Galactosidase (a plasmid having a β-galactosidase gene as a standard for constantly adjusting the efficiency of transfection), 0.2 μg of sirtuin 1 (SIRT1), 0.3 μg of pCMV-β-galactosidase (Myc expression vector or empty vector) for 30 seconds, and the mixture was allowed to stand at room temperature for another 15 minutes. The mixture of Lipopectamine and each vector (or DNA-plasmid) and HEK293 cells were slowly mixed and cultured in a suspension state at room temperature for 20 minutes, then in a 60 mm culture dish and cultured in a CO ₂ incubator for 24 hours. Thereafter, each of the compounds 1 to 5 was added to the concentration as indicated in FIG. 3 on the following day, and then cultured for another 24 hours. Next, the cultured HEK293 cells were washed twice with PBS, and 500 μl of the extraction solution was added to each culture vessel to destroy the cells, followed by centrifugation at 14,000 rpm for 5 minutes. The supernatant (cell extract) thus obtained was used to measure the activity of luciferase and β-galactosidase.

The luciferase activity was determined by incubating 100 μl of the supernatant (cell extract), 100 μl of 20 nM D-luciferin and 300 μl of reaction solution (20 mM glycin, 12.5 mM MgSO ₄ , 3 mM EGTA, 15 mM potassium phosphate, 1 mM DTT , 1 mM ATP) was measured with a luminometer. The β-galactosidase activity was measured to correct the luciferase activity in order to correct the gene injection efficiency. The activity of? -galactosidase was determined by adding 30 μl of the cell extract, 66 μl of ONPT (4 mg / ml), 204 μl of reaction solution (0.1 M sodium phosphate, 1 mM MgCl ₂ , 45 mM β-mercaptoethanol, After reacting in a 37 ° C thermostat, the absorbance was measured with a spectrophotometer. 3) in which only p53-Luc and sirtuin 1 and compound are not added (the first line in Fig. 3), only p53-Luc is added and sirtuin 1 and no compound (The third line of Fig. 3) was treated with DMSO instead of the compound (the same volume as the compound treatment group, 0.1 of the cell culture medium), and the control (p53-Luc and sirtuin 1, % Within).

FIG. 3 shows the results of the above-described luciferase activity of p53. When p53 (p53-luc) was transfected, p53 activity was increased and p53 (p53-luc) and sirtuin 1 (SIRT1) The results showed that the transcriptional activity of p53 was decreased when sialon-1 and p53 were selected.

At this time, it was confirmed that the activity of p53 was further reduced upon treatment with resveratrol, which is a sirtuin 1 activating substance. It is also found that the compounds 1 to 5 also reduce the activity of p53 in cells as well. Therefore, it can be confirmed from the above results that the compounds 1 to 5 have an effect of increasing the activity of sirtuin 1 enzyme.

&Lt; Example 6: NAD ⁺ / Determination of NADH ratio>

It was anticipated that the change in enzyme activity of sirtuin 1 would alter the ratio of NAD ⁺ / NADH in the cells and their concentration in the cells was measured. CYR-to-1 enzyme has been known as a kind of ^'+ NAD-dependent histone energy is activated when the run out state on the cell deacetylase ^{(NAD + dependant histone deacetylase)'} . Therefore, if the ratio of NAD ⁺ / NADH is measured after treatment of a compound in a cell, the degree of activation of sirtuin 1 in the cell can be confirmed.

After culture for HEK293 cells with 70% saturated compounds 1 ~ 5 (2.5 ~ 20㎍ / ㎖) or resveratrol of 4.6㎍ / ㎖ (20μM) was added (molecular weight 228, positive control) and incubated again for 12 hours . Then, HEK293 cells were harvested using PBS and centrifuged at 8,000 rpm for 5 minutes to remove the supernatant. NAD ⁺ / NADH lysis buffer (AAT Bioquesr Cat. 15263) of AAT Bioquest was used to obtain HEK293T cell line, and NAD ⁺ / NADH ratio was increased using AAT Bioquest's NAD ⁺ / NADH Quantitation kit And the results are shown in FIG. 4 (the result 2 means that the content of NAD ⁺ is doubled).

Referring to FIG. 4, it can be seen that the ratio of NAD ⁺ / NADH in the cells is greatly changed by the treatment of the compounds 1 to 5 of the present invention. Compounds 1 to 5 of the present invention increase the ratio of NAD ⁺ / NADH in the cells, and therefore, it can be confirmed that all the compounds derived from these ginsengs are sirtuin-activating compounds.

In particular, Compounds 4 and 5 strongly increased the NAD ⁺ / NADH ratio at a treatment concentration of 2.5 to 5 μg / ml, and Compound 4 strongly increased the NAD ⁺ / NADH ratio, do.

Example 7 Preparation of Young and Aged Cells and Treatment of Samples [

Primary human fibroblasts were isolated from the penis foreskin of newborn infants. Cells were maintained in subculture in DMEM (Dulbecco's Modified Eagles Medium) containing 10% FBS (fetal bovine serum) and 1% antibiotic. The difference in constituent molecular species was observed in young cells (passage 8, p8, 8 passaged cells) with a population doubling of 20 or less and aged cells (passage 35, p35, 35 passaged cells) . Characteristics of senescent cells are characterized by morphological changes, increased beta -galactosidase activity, and reduced rates of cell division. For this purpose, cells were grown in DMEM (Dulbecco's Modified Eagles Medium) for 1 to 2 days to fill 60-70% of cells, and then incubated for 2 days in 0.1% BSA (bovine serum albumin) or serum deficient medium for serum-starvation after the cell, which leads to the moving picture cells in G ₁ stop (arrest G _1), was conducted an experiment for confirmation of the aged cells (Biochem Biophys Res Commun., 302 (4), 778-784, 2003) by. The change of? -Galactosidase in aged cells after the compound treatment was measured using a Cell Signaling dyeing kit (Senescence? -Galactosidase Staining Kit, # 9860).

The human fibroblast (HDF) used in the experiment was prepared by preparing young cells (No. 7 lineages) and aged cells (line No. 35 cells), dividing them in 6-well plates and culturing them in CO ₂ incubator for 48 hours . Compound 4 was dissolved in dimethylsulfoxide (DMSO) to prepare a 1 M stock solution, and the concentration was adjusted to the volume of cell culture medium (DMSO 0.1% or less). The cells were treated with serum free media for 1 day prior to treatment with Compound 4 in aged cell culture to allow for synchronization of the cells. Compound 4 was treated with a micropipette to observe the morphological change after 12 hours through the result of β-galactosidase staining.

On the other hand, the method of staining with? -Galactosidase is as follows. The aging cells were compared using human fibroblasts treated with the compound and a method of staining aged and young cells not treated with the compound as a positive control. Each human fibroblast was washed twice with PBS, 1 ml of fixative solution (1x fixative solution) in the dyeing kit was added to each well, and the mixture was allowed to stand at room temperature for 15 minutes. Each of the cells was washed twice with PBS, and 1 ml of a β-galactosidase staining solution in a dyeing kit was added to each well. The cells were stained for 12 hours, Morphological changes were observed through the staining of β-galactosidase. FIG. 5 shows the result of treating Compound 4 of the present invention at a concentration of 5 μg / ml.

5, the number of aged cells, that is, the number of cells stained with? -Galactosidase, was measured. As a result, aged cells of p35 generation had 70 of 74 cells stained, Two cells were stained. Compound 4 was treated with aging p35 cells. As a result, 36 cells out of 63 cells were stained and the aged cells were transformed into young cells.

<Example 8> Toxicity test>

Example 8-1. Acute toxicity

The toxicity of the butanol fraction and compound 4 of the 90% ethanol extract of ginseng of the present invention to the animal body in an acute (within 24 hours) when the excessive amount of the butanol fraction and the compound 4 were consumed in a short period was investigated and the mortality was determined. Twenty mice of the general mouse ICR mouse line were assigned to each of the control and experimental groups. The control group, PEG-400 / tween-80 / ethanol (8/1/1, v / v / v ) administration only, and the test group are PEG-400 / tween-80 4-butanol fraction, and compound of 90% ethanol extract of Ginseng / Ethanol (8/1/1, v / v / v) and administered orally at a concentration of 100-fold (2 g / kg / day) of 20 mg / kg / day, After 24 hours of administration, the mice were found to survive in the control group and in the experimental group administered with 90% ethanol extract of ginseng and compound 4 at a concentration of 2 g / kg / day.

Example 8-2. Organ organs toxicity test in experimental group and control group

In the long-term toxicity test, the Butanol fraction of the 90% ethanol aqueous solution extract of Ginseng and Compound 4 of the present invention were administered to C57BL / 6J mice (10 rats per group) used as an aging model at 2 g / kg / day for 8 weeks. Administration of butanol fraction, and the compound 4 in 90% ethanol extract of ginseng In order to examine the effect of each organ (tissue) of the animal test and PEG-400 / tween-80 / ethanol (8/1/1, v / v / v), the blood concentration of glutamate-pyruvate transferase (GPT) and blood urea nitrogen (BUN) was measured using a Select E (Vital Scientific NV, Netherland) instrument Respectively. 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

Compound 4 (200 g) of the present invention was 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

Compound 1 (1 g) 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

The butanol fraction of the ginseng 90% ethanol aqueous solution extract of the present invention was added to the cooking seasoning to 1% by weight to prepare a cooking sauce for health promotion.

Formulation Example 2-2. Manufacture of flour food products

The butanol fraction of the ginseng 90% ethanol aqueous solution extract of the present invention was added to the flour at 0.1 wt%, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare a health improving food.

Preparation Example 2-3. Manufacture of soups and gravies

The butanol fraction of the 90% ethanol aqueous solution of ginseng of the present invention was added to the soup and juice at 0.1 wt% to prepare soup for health promotion and juice.

Formulation Example 2-4. Manufacture of dairy products

The butanol fraction of the ginseng 90% ethanol aqueous solution extract of the present invention 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

The vegetable juice for health promotion was prepared by adding the butanol fraction of the 90% aqueous ethanol solution of ginseng of the present invention to 1,000 ml of tomato juice or carrot juice.

Formulation Example 2-6. Manufacture of fruit juice

Healthy fruit juice was prepared by adding 0.1 g of the butanol fraction of the 90% ethanol aqueous solution of ginseng of the present invention to 1,000 ml of apple juice or grape juice.

Claims (10)

( 1 ), at least one ginsenoside selected from the group consisting of Panaxol A (Compound 1 ), Panaxol B (Compound 2 ) and Panaxol C (Compound 3 ) doing,
Characterized in that it comprises a ginseng fraction.
A neurodegenerative disorder, neuropsychiatric disorder, autism, mental retardation, seizure, impaired reflexes, autonomic ataxia, migraine, neuralgia, neuropsychiatric disorders, neuropsychiatric disorders, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment and prophylaxis of pain, liver failure, intestinal obstruction, irritable bowel syndrome, gastroesophageal reflux disease, constipation, diarrhea, renal tubular acidosis, Panconi's syndrome, hypothyroidism, hypothyroidism, hypoparathyroidism, , Ocular optic atrophy, strabismus, retinitis pigmentosa, blindness, hearing loss, degenerative arthritis, rheumatoid arthritis, osteoarthritis, ataxia, dysphagia, dysphagia, stiffness, myopathies, muscular pain, muscular dystrophy, muscle cramps, ptosis Diseases treated by sirtuins activation selected from diseases consisting of muscle paralysis, progressive oculomotor paralysis, lever optic atrophy and respiratory disorders &Lt; / RTI &gt;
[Chemical Formula 1]

The ginseng fraction according to claim 1, wherein the ginseng fraction is obtained by suspending ginseng extract in water by extracting ginseng with water, a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof, Which is a concentrate of an organic solvent layer obtained by adding an organic solvent selected from the group consisting of hexane, ethyl acetate, and butanol,
A neurodegenerative disorder, neuropsychiatric disorder, autism, mental retardation, seizure, impaired reflexes, autonomic ataxia, migraine, neuralgia, neuropsychiatric disorders, neuropsychiatric disorders, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment and prophylaxis of pain, liver failure, intestinal obstruction, irritable bowel syndrome, gastroesophageal reflux disease, constipation, diarrhea, renal tubular acidosis, Panconi's syndrome, hypothyroidism, hypothyroidism, hypoparathyroidism, , Ocular optic atrophy, strabismus, retinitis pigmentosa, blindness, hearing loss, degenerative arthritis, rheumatoid arthritis, osteoarthritis, ataxia, dysphagia, dysphagia, stiffness, myopathies, muscular pain, muscular dystrophy, muscle cramps, ptosis Diseases treated by sirtuins activation selected from diseases consisting of muscle paralysis, progressive oculomotor paralysis, lever optic atrophy and respiratory disorders Or a pharmaceutically acceptable salt thereof. It is a butanol fraction of a concentrate obtained by extracting ginseng with 30 ~ 100% aqueous solution of methanol or ethanol,
(Panaxol A, compound 1 ), paraxol B (compound 2 ), paraxol C (compound 3 ), dimmar-20 (22), 24-diene- β, α 6, β 12 - triol (Dammar-20 (22), 24-diene-3 β, 6 α, 12 β -triol, compound 4), and, 20 (S) - ginsenoside Rg ₃ ( 20 (S ) -Ginsenoside Rg ₃ , Compound 5 ). &Lt; RTI ID = 0.0 &gt; 5. &lt; / RTI &
A neurodegenerative disorder, neuropsychiatric disorder, autism, mental retardation, seizure, impaired reflexes, autonomic ataxia, migraine, neuralgia, neuropsychiatric disorders, neuropsychiatric disorders, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment and prophylaxis of pain, liver failure, intestinal obstruction, irritable bowel syndrome, gastroesophageal reflux disease, constipation, diarrhea, renal tubular acidosis, Panconi's syndrome, hypothyroidism, hypothyroidism, hypoparathyroidism, , Ocular optic atrophy, strabismus, retinitis pigmentosa, blindness, hearing loss, degenerative arthritis, rheumatoid arthritis, osteoarthritis, ataxia, dysphagia, dysphagia, stiffness, myopathies, muscular pain, muscular dystrophy, muscle cramps, ptosis Diseases treated by sirtuins activation selected from diseases consisting of muscle paralysis, progressive oculomotor paralysis, lever optic atrophy and respiratory disorders &Lt; / RTI &gt;

delete delete The present invention includes at least one ginsenoside selected from the group consisting of Panaxol A (Compound 1 ), Panaxol B (Compound 2 ) and Panaxol C (Compound 3 ) doing,
Characterized in that it contains a ginseng fraction,
A neurodegenerative disorder, neuropsychiatric disorder, autism, mental retardation, seizure, impaired reflexes, autonomic ataxia, migraine, neuralgia, neuropsychiatric disorders, neuropsychiatric disorders, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment and prophylaxis of pain, liver failure, intestinal obstruction, irritable bowel syndrome, gastroesophageal reflux disease, constipation, diarrhea, renal tubular acidosis, Panconi's syndrome, hypothyroidism, hypothyroidism, hypoparathyroidism, , Ocular optic atrophy, strabismus, retinitis pigmentosa, blindness, hearing loss, degenerative arthritis, rheumatoid arthritis, osteoarthritis, ataxia, dysphagia, dysphagia, stiffness, myopathies, muscular pain, muscular dystrophy, muscle cramps, ptosis Diseases treated by sirtuins activation selected from diseases consisting of muscle paralysis, progressive oculomotor paralysis, lever optic atrophy and respiratory disorders For the prevention or amelioration of the health functional food.
[Chemical Formula 1]

Novel compounds having the following chemical structure Panaxol A (Compound 1 )

A novel compound Paraxol B (Panaxol B, compound 2 ) having the following chemical structure:

A novel compound paraxol C (Panaxol C, compound 3 ) having the following chemical structure:

The ginseng extract obtained by extracting and 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. To the suspension, hexane, ethyl acetate, and butanol (Compound 1 ), Panaxol A (Compound 2 ), and Panaxol C (Compound 2 ) were obtained from the concentrate of the organic solvent layer obtained by adding an organic solvent selected from the group consisting of 3 ). &Lt; / RTI &gt;
[Chemical Formula 1]

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