KR20130068887A - Compositions comprising extract of myristica fragrans and lignan compounds isolated therefrom for prevention or treatment of disease through activation of sirtuins - Google Patents

Abstract

PURPOSE: A 10-30% ethanol solution extract of Myristica fragrans or a 2,5-bis-aryl-3,4-dimethyl tetrahydrofuran lignan-based compound isolated from the same which activates sirtuin is provided to be used in a pharmaceutical product, a cosmetic product, and a food for preventing or treating diseases related to life span. CONSTITUTION: A pharmaceutical composition for preventing or treating aging contains 10-30% ethanol solution extract of Myristica fragrans containing 0.5 wt% or less of myristicene and one or more compounds selected among nectandrin A, fragransin C1, verrucosin, sucernetindiol, tetrahydrofuroguaiacin, and nectandrin B of chemical formula 1. The pharmaceutical composition enhances activation of sirtuin.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating diseases mediated by sirtuin activation, which comprises a nutmeg extract or a lignan compound isolated therefrom, and a composition for preventing or treating diseases mediated by sirtuin activation }

The present invention relates to a nutritional supplement ( Myristica fragrans extract or a lignan compound isolated therefrom, and more particularly to a composition for preventing or treating diseases mediated by sirtuins activation comprising 10 to 30% ethanol aqueous solution extract of nutmeg or A composition for preventing or treating aging which contains 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compounds separated from the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compounds .

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 senescence-related gerunds 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, cell growth, and cell survival. Among them, p53 regulates apoptosis, FOXO regulates cell survival, PGC-1α regulates glucose hemeostasis, (Bethesda, 21, 404-410, 2006), which has been reported to regulate the activity of these proteins by removing the acetyl group from the peroxisome proliferating activated receptor, gamma, coactivator 1 alpha and the transcription factor p300. Also, as an example of the activity of sirtuin associated with aging, when resveratrol (known to activate sirtuin protein) is administered to yeast, Drosophila, C. elegans, etc. , And extended life span by up to 59% (Nature, 425 (6954), 191-196, 2003). 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. On the other hand, more important than inhibiting aging is the quality of life associated with the reduction of geriatric diseases (Cell, 127 (6), 1109-1122, 2006). Thus, And it has been reported that it is possible to improve the quality of life by significantly reducing adult diseases related to diabetes, heart disease, fatty liver and the like, and sirtuin is becoming an important gene related to aging suppression and healthy life.

The nutmeg ( Myristica fragrans ) is an evergreen tree belonging to the nutmeg family and is a migratory plant grown in Sumatra or Java. Fruits are egg-shaped flesh, and include seeds surrounded by a red scarlet seed coat in the center. Nutmeg has been widely used as a spice in foods such as sauces for a long time and was used as a directional food such as diarrhea, abdominal distension, vomiting and edible decay. However, in addition to the usefulness of the nutmeg, many references have reported that acute toxicity occurs when a large amount of nutmeg is taken. In the nutmeg extract, the alkylbenzene derivative myristate It is known that myristicin, elemicin, and safrole are extracted together.

These compounds are known to exhibit toxic effects similar to those of antipsychotic drugs when they are ingested by humans and are converted into amphetamine derivatives in the body. Traditionally, to remove the toxicity of nutmeg, the nutmeg is soaked in lime for one day, It was also used in medicinal materials. The most abundant toxic substance of nutmeg was Yagaku Zasshi, 128 (1), 129-133, 2008), and the methanol extract of nutmeg has been reported to contain an average of 2.1% myristidine Natural Toxins, 5, 186-192, 1997; Journal of the Korean Pharmacopoeia, 38 (1), 19-21, 2007).

On the other hand, in the present invention, the nutmeg extract containing a large amount of 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound with a small amount of myristidine and a large amount of 2,5- It is confirmed that the activation of the sirtuin enzyme has an effect of preventing or treating diseases associated with aging, diabetes, neurodegenerative diseases, or various diseases associated with abnormal actions of mitochondria, that is, diseases associated with an increase in cell longevity. Diseases associated with abnormal actions of the mitochondria include, but are not limited to, neurological and musculoskeletal disorders and metabolic diseases, which are also associated with 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 visceral 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 diseases include one or more of pancreatic exocrine insufficiency, hypothyroidism, hypoparathyroidism, hypogonadism, developmental delay, type 2 diabetes and hypoglycemia, but are limited thereto 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.

On the other hand, the prior patents relating to the nutmeg extract include "a composition for inhibiting cholesterol ester formation including a herbal extract (Korean Patent No. 399529)", "a pharmaceutical composition for preventing or treating diabetes containing an extract of herb medicine as an active ingredient (Korean Patent No. 793204) " for the prevention or treatment of coronary artery disease or atherosclerosis containing the extract of Chinese medicine as an active ingredient, etc., And the activity of the compound contained in the nutmeg extract is not closely related to the present invention.

The prior patents related to the compounds isolated from the extract of nutmeg include "inhibitor of toxicity caused by anticancer agent and anticancer composition containing it (Korean Patent No. 646574)", "treatment or prevention of acne containing lignan compound as active ingredient , A pharmaceutical composition for the treatment or prevention of an inflammatory disease containing a lignan compound (Korean Patent Registration No. 567431), a medicinal composition for treating liver diseases and liver diseases (Korean Patent No. 619498) Korean Patent No. 579752), "" Pharmaceutical composition for the treatment or prevention of type 2 diabetes (Korean Patent No. 627643) "," PPAR alpha containing the macelignan compound or its pharmaceutically acceptable salt as an active ingredient (Korean Patent No. 830192) ", "a composition for the treatment or prevention of cranial nerve diseases containing lignan compounds ", " (Korean Patent No. 679306), which discloses various activities using a macelignan compound, wherein the macelignan compound is 2,5-bis-aryl-3,4-dimethyl And its structure and properties are different from those of the tetrahydrofuran lignan compound.

In addition, prior patents relating to the activity of the sirtuin include "a composition containing a mulberry extract (Korean Patent No. 826209)", "thiazolopyridine sirtuin regulating compound (Korean Patent Publication No. 2011-0110194)", , "Phthalazinone and related analogues as a sirtuin modulator (Korean Patent Publication No. 2011-0102468) "," isoindolinone and related analogs as a sirtuin modulating agent (Korean Patent Publication No. 2011-0098789) Quinazolinone, quinolone and related analogues (Korean Patent Publication No. 2011-0065536) as a sirtuin modulator, pyridine, bicyclic pyridine and related analogs (Korean Patent Publication No. 2011-0079763) (Korean Patent Publication No. 2011-0063573) as a sirtuin modulator (Korean Patent Publication No. 2011-0063573), benzoxazole, benzthiazole and related analogues as a sirtuin modulating agent (Korean Patent Publication No. 2011-0044291) However, the compounds disclosed in the above patent Name 2,5-bis-aryl-3,4-dimethyl-tetrahydrofuran lignan compound and is different in its structure and properties.

It is an object of the present invention to provide a composition for preventing or treating a disease mediated by sirtuins activation containing a Myristica fragrans extract or a lignan compound isolated therefrom. It is still another object of the present invention to provide an aqueous solution of 10 to 30% ethanol of nutmeg or a 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound (2,5-bis-aryl- , 4-dimethyltetrahydrofuran lignan compounds) as an active ingredient. The present invention also provides a composition for preventing or treating aging.

The present invention relates to a nutritional supplement ( Myristica fragrans extract or a lignan compound isolated therefrom, which comprises administering to a subject in need thereof a therapeutically effective amount of a compound of the invention.

The nutmeg extract or the lignan compound isolated therefrom acts on the activation of the sirtuin, thereby preventing or preventing various diseases which are associated with an increase in cell longevity, such as aging, diabetes, neurodegenerative diseases and abnormal actions of mitochondria, The composition may be used as a therapeutic composition, preferably as a composition for preventing or treating aging. In addition, the nutmeg extract is prepared by extracting nutmeg with a 10-30% ethanol aqueous solution and removing toxic substances such as myristidine and thus has high stability.

Accordingly, preferably, the present invention provides a 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound (2,5-bis- Nectandrin B, Nectandrin A, Fragransin C1, Verrucosin, Saucernetindiol, which are aryl-3,4-dimethyltetrahydrofuran lignan compounds, And at least one compound selected from the group consisting of tetrahydrofuroguaiacin. The present invention also relates to a pharmaceutical composition for preventing or treating aging, which comprises an extract of aqueous ethanol solution of 10 to 30% of nutmeg.

[Formula 1]

The present invention also relates to 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compounds (2,5-bis-aryl-3 Nectandrin B, Nectandrin A, Fragransin C1, Verrucosin, Saucernetindiol and tetrahydrofuran, which are 4-dimethyltetrahydrofuran lignan compounds. The present invention relates to a pharmaceutical composition for preventing or treating aging, which comprises at least one compound selected from the group consisting of Tetrahydrofuroguaiacin.

The 2,5-bisaryltetrahydrofuran lignan compound is obtained by grinding nutmeg and extracting it with 10 to 30% aqueous solution of ethanol; And purely separating and purifying 2,5-bisaryltetrahydrofuran lignan compound having a sirtuin-activating effect using chromatography.

The 10 to 30% ethanol aqueous solution extract of the nutmeg may further be used to remove the remaining trace amount of myristidine and concentrate the 2,5-bisaryltetrahydrofuran lignan compound using an ion exchange resin. Thus, the ion-exchange resin eluate can be used as an extract of aqueous solution of 10 to 30% ethanol in nutmeg, such as Nectandrin B, Nectandrin A, which is a 2,5-bisaryltetrahydrofuran lignan compound of Formula 1, A compound containing at least one compound selected from the group consisting of a compound of the formula (I), a compound selected from the group consisting of Fragransin C1, Verrucosin, Saucernetindiol and Tetrahydrofuroguaiacin, . In addition, the myristidine content of the ion exchange resin eluate of the nutmeg can be 0.1% by weight or less. More specifically, the ion exchange resin may be one of aromatic non-substituted resins (Diaion HP-20, SP825, AXT204, XAD1600T, and MN200) as a synthetic adsorbent.

Nectandrin B, Nectandrin A, Fragransin C1, Verrucosin, and Suchenetin, which are 2,5-bisaryltetrahydrofuran lignan compounds of the present invention, Saucernetindiol and Tetrahydrofuroguaiacin can be separated and extracted from nutmeg by extraction with an organic solvent (alcohol, ether, acetone, etc.), hexane and water, and column chromatography. Known methods used can be easily obtained by using alone or in suitable combination. The crude extract can be further purified according to the conventional method, if necessary.

The chromatography used in the present invention includes 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.

In addition, the 10 to 30% ethanol aqueous solution extract of the nutmeg of the present invention or the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound isolated therefrom not only can be easily separated from nutmeg, Therefore, it can be used as an additive for foods and medicines.

The pharmaceutical composition comprising the aqueous solution of 10 to 30% ethanol in nutmeg or the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound isolated therefrom may be formulated into powders, granules, And may be formulated in the form of tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories, and sterilized injection solutions. 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 be prepared by mixing the aqueous solution of 10 to 30% ethanol in the nutmeg of the present invention or the 2,5-bis- Dimethyltetrahydrofuran lignan compound is prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin and the like. 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. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The dosage of the pharmaceutical composition containing the 10 to 30% ethanol aqueous solution extract of the nutmeg or the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound isolated therefrom of the present invention may vary depending on the age, Sex, weight and the particular disease or condition being treated, the severity of the disease or condition, the route of administration, and 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 pharmaceutical composition containing the extract of aqueous solution of 10 to 30% ethanol in nutmeg or the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound isolated therefrom of the present invention can be administered to mammals such as rats, ≪ / RTI > by 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 10 to 30% ethanol aqueous solution extract of nutmeg and a food acceptable food supplementary additive. 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 treatment of diseases mediated by sirtuin activation, including aging, diabetes, neurodegenerative diseases, and abnormalities of mitochondria, including 10-30% aqueous ethanolic extracts of nutmeg and pharmaceutically acceptable food- A health functional food for preventing or ameliorating a disease associated with the action.

The concentration of 2,5-bisaryltetrahydrofuran lignan compound in the 10-30% aqueous ethanol extract of nutmeg according to the present invention is lower than that of the toxic substance myristicine, It is confirmed that the activity effect is high. In addition, the extract of aqueous solution of 10 to 30% ethanol of nutmeg or the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound isolated therefrom has an effect of activating sirtuins enzyme, Through the action on energy metabolism, medicines, cosmetics and foods for the prevention or treatment of various diseases related to aging, diabetes, neurodegenerative diseases and abnormal actions of mitochondria, Can be usefully used.

FIG. 1 is a graph showing the spectral results of HPLC analysis of 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compounds separated from an extract obtained by extracting nutmeg with 30% ethanol.
FIG. 2 is a graph showing the result of Western blotting that the phosphorylation of p53 and the expression of p21 decrease to the level of young cells after treatment with nethendrine B (10-20 μM) in senescent cells to be.
FIG. 3 is a graph showing the results showing that the individual survival rate of the fruit fly treated with the above-mentioned nectandrin B was higher than that of the control by administering the nectandrin B (50 μg / day) to the fruit fly.
Figure 4 shows that nexandrine B (20 mg / kg / day) was administered to naturally aged C57BL / 6J male mice at 96 weeks of age (24 months) And the activity was increased by Western blotting.

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 scope of the invention to those skilled in the art.

< Example  One : Myristicine  Low in content nutmeg  Preparation of ethanol aqueous solution extract &gt;

100 g of crushed nutmeg was placed in 500 ml of each solvent (see Table 1), and an extract was prepared for each solvent condition using an ultrasonic wave extender three times for 2 hours. The extracted extracts were used at the same concentration and the Myristicine standard was purchased from Sigma (product number: M9237). Each nutmeg extract and Myristicin standards were analyzed by HPLC (Optima Pak C ₁₈ column, 4.6 x 250 mm, 5 μm particle size) under conditions of MeOH-H ₂ O (0-32 min: 63% MeOH, 32-37 min: 63 → 100% MeOH) Size, flow rate 1 ml / min, UV detection: 260 nm]. The mysticin content of each nutmeg extract is shown in Table 1.

Alcohol extraction solvent Among the extracts
Myristicin content (%) Among the extracts
Nectarine B content (%) Water extract 0.19% 0.62 10% aqueous solution of ethanol 0.31% 0.94 20% ethanol aqueous solution extract 0.33% 2.98 30% ethanol aqueous solution extract 0.45% 7.48 40% ethanol aqueous solution extract 1.34% 1.95 50% ethanol aqueous solution extract 1.48% 2.77 75% Ethanol Aqueous Extract 1.27% 2.95 75% aqueous methanol solution extract 1.85% 7.47

As shown in Table 1, it was confirmed that the myristidine content of the 30% ethanol aqueous solution extract was 3 times smaller than that of the extract of other conditions. The nutmeg extract of each condition was further purified by HPLC (OptimaPak_C ₁₈ column 4.6 x 250 mm, 5 μm particle size, flow rate 1 ml) under conditions of MeOH-H ₂ O (0-35 min: 60% MeOH, 35-60 min: 60 → 100% MeOH) / min, UV detection: 205, 280 nm). As a result, it was found that nectandrin B was most extracted from the 30% ethanol aqueous solution extract (see Table 1). Therefore, it was confirmed that the nutmeg extract was extracted with an ethanol aqueous solution of 30% or less while maximizing the amount of the active substance and minimizing the toxic substance content.

Example 2: Additional removal of toxic substance myristicine from aqueous extract of aqueous solution of nutmeg ethanol>

100 g of nutmeg was extracted with 500 ml of a 30% aqueous ethanol solution and then adsorbed through 500 g of Diaion HP-20, one of ion exchange resins. Then, the adsorbate was eluted with 500 ml each of 30 to 90% ethanol aqueous solution, 100% ethanol and 100% acetone, and the contents of myristidine and nectandrin B contained in the eluate were shown in Table 2. Referring to Table 2, it was confirmed that myristicine was hardly detected when the adsorbate was eluted with an ethanol aqueous solution of 80% or less. In addition, it was confirmed that the use of 80% ethanol aqueous solution as the solvent elicited a large amount of nethandrine B.

Eluting solvent condition 30% aqueous ethanol solution 50% aqueous ethanol solution 70% ethanol aqueous solution 80% aqueous ethanol solution 90% aqueous ethanol solution 100% ethanol Acetone The content of nectandrin B in the eluate 0.01%   0.1% 24.2%   61.0%   13.9% 1.0%    3.0% The content of myristidine in the eluate 0.04% 0.04% 0.04% 0.04% 0.66% 20.77% 13.71%

< Example  3: From nutmeg  Identification of extracted compounds &gt;

The 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound was isolated from the nutmeg extract of 30% aqueous ethanol solution of Example 1 by HPLC, and the compound was analyzed by HPLC (see FIG. 1) ¹ H, and ¹³ C-NMR. The physicochemical properties of the compound are as follows.

3-1. Tetrahydrofuroguanidine  ( Tetrahydrofuroguaiacin , Compound 1)

As a colorless powder, hydrogen nuclear magnetic resonance _{spectrum: ppm (500 MHz, CDCl 3} ): δ 0.61 (6H, d, J = 6.0 Hz, 3- and 4-Me), 2.67 (2H, m, 3- and 4- H), 3.91 (6H, 3'- and 3'-OMe), 5.12 (2H, d, J = 6.6 Hz, 2- and 5-H) ), 6.90-6.99 (6H, m, 2'-, 5'-, 6'-, 2 "-, 5" - and 6 "-H), carbon nuclear magnetic resonance _{spectrum: ppm (125 MHz, CDCl 3} ) C-2 and C-4), 55.8 (2xMe), 82.7 (C-2 and C-5), 109.0 2 "), 113.9 (C-5 'and C-5"), 119.3 (C-6' and C- C-4 "), 146.2 (C-3 'and C-3").

3-2. Sausage net dior  ( Saucernetindiol , Compound 2)

As a colorless oil, hydrogen nuclear magnetic resonance _{spectrum: ppm (500 MHz, CDCl 3} ): δ 0.63 (3H, d, J = 6.5 Hz, 4-Me), 1.01 (3H, d, J = 6.5 Hz, 3-Me ), 2.33 (2H, m, 3- and 4-H), 3.90-3.92 (6H, 3'- and 3'-OMe), 4.65 (1H, d, J = 9.5 Hz, 1H, d, J = 4.5 Hz, 5-H), 5.58 (2H, s, 4'- and 4'-OH), 6.77-6.99 (6H, m, 2 "-, 5" - and 6 "-H), carbon nuclear magnetic resonance _{spectrum: ppm (125 MHz, CDCl 3} ): δ 9.42 (3-Me), 11.83 (4-Me), 43.4 (C-3) (C-4), 55.8 (2xMe), 84.8 (C-2), 85.7 (C-5), 108.3 ), 114.0 (C-5), 118.8 (C-6), 119.3 (C-6) , 145.0 (C-4 "), 146.3 (C-3"), 146.6 (C-3 ').

3-3. Berukosin  ( Verrucosin , Compound 3)

As a colorless oil, hydrogen nuclear magnetic resonance _{spectrum: ppm (500 MHz, CDCl 3} ): δ 0.67 (3H, d, J = 6.5 Hz, 4-Me), 1.06 (3H, d, J = 6.5 Hz, 3-Me ), 1.79 (1H, m, 3-H), 2.25 (1H, m, 4-H), 3.87-3.89 (6H, 3'- and 3 "-OMe), 4.36 (1H, d, J = 9.5 Hz , 2'-, 5'-, 6'-, 2'-, 5''- and 2'H), 5.10 (1H, d, J = 9.0Hz, 5H), 6.80-6.96 6 "-H), Carbon Nuclear Magnetic Resonance Spectrum: ppm (125 MHz, CDCl ₃ ):? 14.8 (3-Me), 15.2 (4-Me), 45.9 C-2 '), 111.6 (C-2'), 115.7 (C-5 '), 116.1 (C- C-4 '', 146.8 (C-4 ''), 120.7 (C-6 ' , 148.6 (C-3 "), 149.0 (C-3 &apos;).

3-4. Nectandrine  B ( Nectandrin  B, compound 4)

(600 MHz, CDCl ₃ ):? 1.05 (6H, d, J = 6.0 Hz, 3- and 4-Me), 2.35 H), 3.85 (6H, 3'- and 3 "-OMe), 4.53 (2H, d, J = 5.4 Hz, 2- and 5-H), 5.74 (2H, brs, 4'- and 4" -OH ), 6.91 (2H, d, J = 7.8Hz, 5'- and 5''H), 6.93 (2H, dd, J = 1.8, 7.8Hz, d, J = 1.8 Hz, 2'- and 2 "-H), carbon nuclear magnetic resonance _{spectrum: ppm (200 MHz, CDCl 3} ): δ 133.9 (C-1 'and C-1"), 114,1 ( C-2 'and C-2 "), 146.4 (C-3' and C-3"), 144.9 (C-6 and C-6), 87.2 (C-2 and C-5), 44.1 (C-3 and C-4), 12.7 × 2).

3-5. Nectandrine  A ( Nectandrin  A, compound 5)

White crystals, hydrogen nuclear magnetic resonance _{spectrum: ppm (500 MHz, CDCL 3} ) δ: 1.00 (3H, d, J = 3.5 Hz, 4-Me), 1.02 (3H, d, J = 3.5 Hz, 3-Me) (1H, d, J = 7.5 Hz, 2-H), 2.27 (2H, m, 3- and 4-H), 3.80-3.86 J = 7.5 Hz, 5-H), 6.81-7.09 (6H, m, Ar-H); Carbon Nuclear Magnetic Resonance Spectrum: ppm (125 MHz, CDCl ₃ )?: 13.1 (3-Me), 13.2 (4-Me), 45.6 (C-4), 45.5 OMe), 88.0 (C-5), 88.2 (C-2), 110.9 (C-2 '), 111.3 (C-6), 120.0 (C-6), 135.1 (C-1 '), 136.4 -3 "), 150.4 (C-3 &apos;).

3-6. Praagan Shin C1  ( Fragrance C1 , Compound 6)

As a colorless oil, hydrogen nuclear magnetic resonance _{spectrum: ppm (500 MHz, CDCl 3} ): δ 1.04 (3H, d, J = 6.6 Hz, 3-Me), 1.06 (3H, d, J = 7.2 Hz, 4-H ), 2.32 (1H, m, 3-H), 2.34 (1H, m, 4-H), 3.88 (9H, 3'-, 5'- and 3 "-OMe), 4.50 (1H, d, J = 7.2 Hz, 2H), 4.52 ( 1H, d, J = 7.2 Hz, 5-H), 5.47-5.58 (2H, br, s, 4'- and 4 "-OH), 6.67 (2H, br, s, 2'- and 6'-H) , 6.91-6.97 (3H, m, 2 "-, 5" - and 6 "-H), carbon nuclear magnetic resonance _{spectrum: ppm (125 MHz, CDCl 3} ): δ (C-3), 44.5 (C-4), 56.3-55.8 (3xMe), 87.2 (C-2), 87.5 , 103.1 (C-2 'and C-6'), 102.9 (C-2 "), 114.1 1 "), 145.1 (C-4 '), 146.4 (C-4"), 146.9 (C-3', C-5 'and C-3 ").

< Example  4: Preparation of young and senescent cells>

The young and senescent cells used in the present invention were prepared using human fibroblasts (primary cells). The cells were separated from the penile foreskin of newborn infants and subcultured with trypsin-EDTA in DMEM (Dulbecco's Modified Eagles Medium) containing 10% fetal bovine serum (FBS) and 1% Cells were cultured. Of these, younger cells were used to carry out subculture for 10 times, and aged cells were used for subculture 30 times or more.

< Example  5: Siruin  Enzyme activity confirmation>

To confirm the effect of the extract of aqueous solution of 10 to 30% ethanol of nutmeg of the present invention or the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound separated therefrom on the activity of the sirtuin enzyme The expression levels of Weisurufin 1 to 3 were confirmed.

To this end, young cells (subculture 8 times) and aged cells (subculture 35 times) were cultured on a cell culture dish (60 mm) to fill 60-70% of the bottom of the culture dish ), 0.1% BSA (bovine serum albumin) or serum-starvation medium for 2 days to reach G1 arrest state. Thereafter, in the exemplary cell of the example 30% ethanol aqueous solution of 1 nutmeg extract (20 ~ 40㎍ / ㎖), the second embodiment of the 80% ethanol eluent (20 ~ 40㎍ / ㎖), 1 to a compound of the invention 6 (10-20 μM). The compounds 1 to 6 were dissolved in dimethylsulfoxide (DMSO) to prepare a 1 M stock solution, which was diluted in a cell culture medium.

Western blot analysis was performed to confirm the amount of protein expression. Each cell was treated with SDS sample buffer, and the cells were disrupted using ultrasound to obtain a protein eluate. Thereafter, 10% SDS-PAGE electrophoresis was performed using the protein eluate, and the protein was fixed on a nitrocellulose transfer membrane using a wet transfer kit. The membrane was blocked with 5% skim milk for 1 hour at room temperature, reacted with a primary antibody and a secondary antibody against each protein, and then the change in the expression level of each protein was measured The results are shown in Table 3. Each result was expressed as a fold value for the control group of the aged cells as a reference (1.00).

Aging cells (subculture 35 times) Protein expression (fold) Sylt one 1 Sylt 2 Sir Too 3 Control group density 1.0 ± 0.1 1.0 ± 0.1 1.0 ± 0.1 Compound 1 (tetrahydrofuroguiaacin) -treated group 10 μM 2.5 ± 0.2 1.5 ± 0.2 2.5 ± 0.1 20 μM 4.3 ± 0.2 2.8 ± 0.1 4.7 ± 0.1 Compound 2 (saponin-diol) treated group 10 μM 3.8 ± 0.3 1.3 ± 0.1 3.8 ± 0.2 20 μM 5.4 ± 0.1 2.5 ± 0.2 5.8 ± 0.1 Compound 3 (berukosin) treated group 10 μM 2.9 ± 0.2 1.2 ± 0.2 3.9 ± 0.2 20 μM 4.6 ± 0.1 2.3 ± 0.3 5.6 ± 0.2 Compound 4 (nectandrin B) treated group 10 μM 4.4 ± 0.2 1.4 ± 0.1 5.3 ± 0.1 20 μM 8.3 ± 0.2 2.3 ± 0.1 6.0 ± 0.2 Compound 5 (nectandrin A) treated group 10 μM 4.3 ± 0.3 1.3 ± 0.1 4.3 ± 0.2 20 μM 6.2 ± 0.2 2.6 ± 0.2 6.3 ± 0.1 Compound 6 (pragrance C1) treated group 10 μM 4.3 ± 0.1 1.2 ± 0.1 4.8 ± 0.2 20 μM 6.5 ± 0.1 2.4 ± 0.2 6.6 ± 0.2 Example 1
30% ethanol aqueous solution extract of nutmeg 20 占 퐂 / ml 2.6 ± 0.2 1.2 ± 0.2 2.5 ± 0.1 40 µg / ml 4.4 ± 0.2 2.5 ± 0.1 4.4 ± 0.1 The 80% ethanol aqueous solution eluate of Example 2 20 占 퐂 / ml 3.4 ± 0.2 1.2 ± 0.1 3.4 ± 0.1 40 µg / ml 5.6 ± 0.1 2.2 ± 0.2 5.4 ± 0.1 The amount of protein expression of sirtuin 1 and 3 was 6 to 7 times higher than that of the control (fold value 1.00) of aged cells (subculture 35 times) in young cells (subculture 8 times) Protein expression level is 2 ~ 3 times higher

Referring to the results in Table 3, it can be seen that the 30% ethanol aqueous solution extract of the nutmeg of Example 1, the 80% ethanol eluate of Example 2, the sirtuin 1 to 3 It was confirmed that the expression level of the protein was remarkably increased compared to the control group. In addition, it was found that the extracts and the compounds had an effect of increasing the expression amount of sirtuin 1 to 3 protein in aging cells in a concentration-dependent manner.

< Example  6: Aging Marker  Expression confirmation>

In order to examine the effect of the compound of the present invention on aging cells, after treatment of aging cells (subculture 35 times) with nethandrine B (10 to 20 μM), phosphorylation of p53 And p21 expression level were confirmed by Western blotting. The western blot was performed in the same manner as in Example 5, and the results are shown in Fig.

2, the expression of p21 and the phosphorylation of p53, which were increased in the aged cells compared with that of young cells (performed 8 times in the subculture), were reduced to the level of young cells after treatment with nectandin B Therefore, it was confirmed that cell proliferation occurs in senescent cells as the cell cycle is activated by treatment with nectandrin B.

< Example  7: In Drosophila  Verification of life extension>

[Step 1] Preparation and breeding of fruit flies

Wild-type Canton-S flies were obtained from the Bloomington Drosophila Stock Center (BDSC) Indiana, USA.

[Step 2] Measuring the effect of extending the life of the fruit flies

The effect of longevity of Drosophila is determined by measuring median life span after 50% reduction of whole Drosophila after adult, where the same amount of PEG (polyethylene glycol) (50 ㎍ / day) was dissolved in PEG, and the survival rate of the fruit flies was shown in Fig.

Referring to FIG. 3, it was found that the survival rate of Drosophila was increased in the group treated with nectandrin B (50 μg / day). As a result, it was found that in the Drosophila individuals treated with nectandrin B, .

< Example  8: Anti-aging effect test using natural aging animals>

[Step 1] Sample preparation

For anti-aging experiments, nectandrin B (20 mg / kg / day) was orally administered to C57BL / 6J male mice once every other day. Nectandrin B was prepared by dissolving in a 30% aqueous solution of polyethylene glycol-400 (Sigma Co., USA).

[Step 2] Establishment of experiment group and verification of effects on aging animals

As aging animals, naturally aged C57BL / 6J male mice at 96 weeks (24 months) were prepared in groups of 10 mice. C57BL / 6J male mice at 6 weeks of age were prepared as control animals. The mice were divided into individual mice and maintained at 12-hour intervals (7:00 am to 17:00 pm) and at night intervals after having a one-week adaptation period.

The test group for the anti-aging effect test was ① 6-week-old mouse normal diet ① 96-week old mouse normal diet ② 96-week old mouse nextandrin B (10 mg / kg / day) ) Treatment group. The nectan green B treatment group orally administered nectandrin B (10 to 20 mg / kg / day) once a day for 12 weeks for a certain period of time (10 am) in addition to the normal diet. After 4 weeks of treatment, each mouse was sacrificed, and the amount of protein expression of sirtuin 1 to 3 was confirmed in muscle, and the results are shown in Fig. In the case of the normal diet, 30% aqueous solution of polyethylene glycol-400 (Sigma Co., USA) was administered instead of nectandrin B.

4, the expression of sirtuin 1 to 3 was significantly reduced in 96-week-old mice (aged animals) than in 6-week-old mice (young animals), whereas nectandrin B In the muscle of 96-week-old mice (aged animals treated with nectandrin B), the expression of sirtuin 1 to 3 was found to increase in a dose-dependent manner. In particular, at a dose of 20 mg / kg / day of nectandrin B, the expression of sirtuin 2 and 3 was increased in a similar manner to that of 6-week-old mice (young animals).

< Experimental Example  9: Toxicity test>

9-1. Acute toxicity

This experiment was conducted to investigate the toxicity of a 10 to 30% aqueous ethanol solution of nutmeg and nexandrine B of the present invention to an animal body in an acute (within 24 hours) when an excessive amount of the extract was administered in a short period of time and to determine the mortality rate . 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 / EtOH (8/1/1, v / v / v) / EtOH (8/1/1, v / v / v) and administered orally at a concentration of 100-fold (2 g / kg / day) of the administration concentration used in Example 8 of 20 mg / kg / day. After 24 hours of administration, the mice were found to survive in the control group and in the test group administered with 2 g / kg / day of nutmeg extract and nectandrin B, respectively.

9-2. Experimental group  And control organ organs and tissue toxicity experiments

The long-term toxicity test was carried out by administering the present invention of the present invention to a C57BL / 6J mouse (10 rats per group) using a 10-30% ethanol aqueous solution extract of the present invention and nextendrine B at various concentrations for 8 weeks. In order to investigate the effects on the organs (tissues) of the animals, the experimental group administered with the aqueous solution of 10 to 30% ethanol aqueous solution of nutmeg and the nextendrine B of the present invention, and PEG-400 / tween-80 / EtOH (8/1/1, After 8 weeks from the control animals receiving only v / v / v, blood was collected and the blood levels of GPT (glutamate-pyruvate transferase) and BUN (blood urea nitrogen) were measured by Select E (Vital Scientific NV, . 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 sampled from each animal, and histological observation was performed with an optical microscope through a conventional tissue section production process. No abnormalities were observed in all tissues.

< Formulation example  1: Pharmaceutical preparations>

1-1. Manufacture of tablets

200 g of nectandrin B 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.

1-2. Injection preparation

1 g of nectandrin B, 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 manufacturing>

2-1. Manufacture of cooking seasonings

The spice for health promotion was prepared by adding 1% by weight of the nutmeg 30% ethanol aqueous solution extract of the present invention to the cooking seasoning.

2-2. Manufacture of flour food products

The nutritional enhancer was prepared by adding 0.1% by weight of the nutmeg 30% ethanol aqueous solution extract of the present invention to wheat flour and preparing bread, cake, cookies, crackers and noodles using the mixture.

2-3. soup  And juicy ( gravies Manufacturing

Health enhancing soup and juice were prepared by adding a 30% ethanol aqueous solution extract of nutmeg according to the present invention to soup and juice at 0.1 wt%.

2-4. dairy product( dairy products Manufacturing

The nutmeg 30% ethanol aqueous solution extract of the present invention was added to milk in an amount of 0.1% by weight and various dairy products such as butter and ice cream were prepared using the milk.

2-5. Vegetable juice  Produce

Healthy vegetable juice was prepared by adding 0.5 g of the nutmeg 30% ethanol aqueous solution extract of the present invention to 1,000 ml of tomato juice or carrot juice.

2-6. Fruit juice  Produce

Health enhancing fruit juice was prepared by adding 0.1 g of the nutmeg 30% ethanol aqueous solution extract of the present invention to 1,000 ml of apple juice or grape juice.

Claims (7)

Myristicin content is 0.5% by weight or less, Nectandrin B (Nectandrin B), Nectandrin A (Fragransin C1), Verrucosin (Verrucosin), Saucerinetinol (Saucernetindiol) and tetrahydrofuroguaiacin (Tetrahydrofuroguaiacin) pharmaceutical composition for the prevention or treatment of aging, characterized in that it contains an extract of 10-30% ethanol aqueous solution containing nutmeg containing at least one compound selected from compounds.
[Formula 1]

The method of claim 1,
The nutmeg 10-30% aqueous solution of ethanol extract, the content of myristicin, which is a toxic substance using an ion exchange resin, 0.1 wt% or less, Nectandrin B of Formula 1, Nectandrin A (Nectandrin) A), fragransin C1 (Fragransin C1), berucosin (Verrucosin), Sausnetinediol (Saucernetindiol) and tetrahydrofuroguaiacin (Tetrahydrofuroguaiacin) is characterized in that it comprises one or more compounds selected from A pharmaceutical composition for preventing or treating aging.
[Formula 1]

3. The method according to claim 1 or 2,
The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of aging, characterized in that for increasing the activity of sirtuins enzyme (sirtuins). Mysticin is a toxic substance is 0.5% by weight or less, Nectandrin B (Nectandrin B), Nectandrin A (Fragransin C1), Verrucosin (Verrucosin), Sau Prevention or improvement of aging, characterized in that it contains an extract of 10-30% ethanol aqueous solution of nutmeg containing at least one compound selected from the group consisting of sunetinediol (Saucernetindiol) and tetrahydrofuroguaiacin (Tetrahydrofuroguaiacin) Health functional food.
[Formula 1]

The method of claim 4, wherein
The health functional food is selected from the group consisting of nutritional products including drinks, meat, sausages, bread, candy, snacks, noodles, dairy products including ice cream, soups, beverages including ionic drinks, alcoholic beverages and vitamin complexes Functional food for the prevention or improvement of aging, characterized in that. Nectandrin B, Nectandrin A, Fragransin C1, Verrucosin, Sauceretinediol and tetrahydrofuroguacin of Formula 1 Tetrahydrofuroguaiacin) pharmaceutical composition for the prevention or treatment of aging, characterized in that it contains at least one compound selected from the group consisting of compounds as an active ingredient.
[Formula 1]

The method according to claim 6,
The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of aging, characterized in that for increasing the activity of sirtuins enzyme (sirtuins).

Images