KR101630820B1 - Pharmaceutical composition for blood vessel disease prevention or treatment comprising substance extracted from Hericium erinacium - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating a vascular disease, which comprises a mushroom-derived material, and more particularly, to a pharmaceutical composition for preventing or treating vascular disease, A pharmaceutical composition for therapeutic use, and a pharmaceutical composition for inhibiting angiogenesis.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing or treating vascular diseases,

The present invention relates to a pharmaceutical composition for preventing or treating a vascular disease, which comprises a mushroom-derived material, and more particularly, to a pharmaceutical composition for preventing or treating vascular disease, A pharmaceutical composition for therapeutic use, and a pharmaceutical composition for inhibiting angiogenesis.

The endothelial cells of the vascular endothelium are advantageous for endothelial cell-derived nitric oxide (NO) and prostanoid, and can activate and degrade vascular activity-related hormones to regulate vascular smooth muscle function, thereby contraction and / Can be relaxed.

The telomerase activity of vascular endothelial cells decreases with aging. In addition, in aging vascular endothelial cells, production of NO, expression of endothelial nitric oxide synthase (eNOS) and production of prostacyclin are decreased, resulting in a remarkably low contractile force of blood vessels. PAI-1 (type I plasminogen activator ) Expression is increased and thrombogenesis is promoted and the expression of cell adhesion proteins and chemokines such as Inter-Cellular Adhesion Molecule 1 (ICAM-1), interleukin-1 and interleukin-8 is increased, . In fact, the vascular endothelial cells of atherosclerotic lesion tissue are known to have increased SA-β-Gal (senescenceassociated beta-galactosidase) activity, which is well known as a cell senescence marker, compared to normal tissue cells. In addition, the cells of atherosclerotic lesions are more likely to have telomere shortening than normal tissue cells. Therefore, inhibition and delay of vascular endothelial cell senescence may prevent and treat cardiovascular diseases.

In addition, in the prior arts disclosed in Japanese Patent Application Laid-Open No. 10-2004-0050396 (published on June 16, 2004), nitrogen monoxide (NO), which comprises at least one member selected from the group consisting of citrine, And a pharmaceutical composition using the same.

Furthermore, the patent document 10-2013-0046115 (published on May 07, 2013) discloses a composition for inhibiting angiogenesis, which comprises siring ginsenoside as an active ingredient, And activating telomerase exhibits excellent anti-aging effects.

In addition, a well known method of delaying blood vessel aging has been calorie restriction. However, this is not easy to apply and it causes discomfort in daily life.

The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a pharmaceutical composition and / or a pharmaceutical composition suitable for use in the prevention or treatment of vascular diseases, which has an effect of inhibiting the aging of blood vessels by inhibiting SA- And to provide a health functional food for preventing or improving vascular diseases.

The present invention provides a pharmaceutical composition for preventing or treating vascular diseases, which comprises a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

According to a preferred embodiment of the present invention, the vascular disease is one or more diseases selected from the group consisting of diabetes, cerebrovascular disorder, cardiovascular disorder, obstructive arteriosclerosis, dementia, atherosclerosis, hypertension and obesity . ≪ / RTI >

According to another preferred embodiment of the present invention, the compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof may be derived from a mushroom.

The present invention also provides a pharmaceutical composition for inhibiting angiogenesis, comprising the compound represented by the above-mentioned formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

According to a preferred embodiment of the present invention, the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof may be one derived from a mushroom of Aspergillus oryzae.

Further, the present invention provides a health functional food for preventing or ameliorating a vascular disease comprising the compound represented by the above-mentioned formula (1) or a salt thereof as an active ingredient.

According to a preferred embodiment of the present invention, the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof may be one derived from a mushroom of Aspergillus oryzae.

In addition, the present invention provides a health functional food for prevention or improvement of vascular aging comprising the compound represented by the formula (1) or a salt thereof as an active ingredient.

According to a preferred embodiment of the present invention, the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof may be one derived from a mushroom of Aspergillus oryzae.

In addition, the present invention relates to a method for producing an extract, which comprises mixing and extracting Cucurbitaceae and a C ₁ -C ₄ alcohol to prepare an extract; Fractionating the extract with an organic solvent to produce a fraction; And chromatographing the fractions to obtain a compound represented by the following formula (1): Wherein the organic solvent comprises at least one member selected from the group consisting of a nucleic acid, methylene chloride, ethyl acetate, and a C ₁ -C ₄ alcohol.

According to a preferred embodiment of the present invention, the organic solvent may include methylene chloride and methanol.

The present invention provides a pharmaceutical composition suitable for use in the prevention or treatment of vascular diseases and / or a health functional food for preventing or ameliorating vascular diseases, which has an excellent anti-aging effect by inhibiting SA-beta-Gal activity, It is effective.

1 shows the ¹ H NMR results of Compound 1 of the present invention.
2 shows the ¹³ C NMR results of the compound 1 of the present invention.
3 shows the results of ESI-MS spectrum analysis of Compound 1 of the present invention.
4 shows IR spectrum analysis results of Compound 1 of the present invention.
Fig. 5 shows the result of measuring cytotoxicity of Compound 1 in Experimental Example 2. Fig.
FIG. 6 shows the results of measuring the anti-angiogenic activity of Compound 1 in Experimental Example 3. FIG.
FIG. 7 shows the results of measuring the inhibitory activity of Compound 1 against fibroblastogenesis in Experimental Example 3. FIG.
Fig. 8 shows the results of measuring the anti-angiogenic activity of Compound 1 in Experimental Example 3. Fig.
FIG. 9 shows the results of measuring the fibroblast aging inhibitory activity of Compound 1 in Experimental Example 3. FIG.

Hereinafter, the present invention will be described in more detail.

As described above, a well-known method for delaying blood vessel aging up to now is calorie restriction. However, this is not easy to apply and it causes discomfort in everyday life. Therefore, another alternative is required.

Accordingly, the present invention has been made to solve the above-mentioned problems by providing a pharmaceutical composition for preventing or treating vascular diseases, which comprises a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. There is an effect of providing a pharmaceutical composition for preventing or treating vascular diseases having an excellent anti-aging effect by inhibiting SA-beta-Gal activity.

[Chemical Formula 1]

The present invention provides a pharmaceutical composition for preventing or treating vascular diseases comprising the compound represented by the above-mentioned formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof is not particularly limited as long as it can be usually prepared and / or purchased, but it may preferably be one which has been liberated from the mushroom.

In addition, the pharmaceutically acceptable salt thereof may include the compound of the formula (1). Further, the pharmaceutical compositions may be lyophilized and include such agents or salts thereof that can be reconstituted to form pharmaceutically acceptable agents for administration, such as by intravenous, intramuscular or subcutaneous injection.

In addition, the vascular disease is not particularly limited as long as it is a disease that can usually be caused by aging and / or a decrease in elasticity of blood vessels. Preferably, the vascular disease is diabetes, cerebrovascular disorder, cardiovascular disorder, Atherosclerosis, hypertension, and obesity. ≪ Desc / Clms Page number 2 >

In addition, the present invention provides a pharmaceutical composition for inhibiting angiogenesis, comprising the compound represented by the above-mentioned formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof is not particularly limited as long as it can be usually prepared and / or purchased, but it may preferably be one which has been liberated from the mushroom.

In addition, the pharmaceutically acceptable salt thereof may include the compound of the formula (1). Further, the pharmaceutical compositions may be lyophilized and include such agents or salts thereof that can be reconstituted to form pharmaceutically acceptable agents for administration, such as by intravenous, intramuscular or subcutaneous injection.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, And the like. When administered orally, the protein or peptide is extinguished and the oral composition should be formulated to coat the active agent or protect it from degradation from above. The pharmaceutical composition may also be administered by any device capable of transferring the active agent to the target cell.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001 to 100 mg / kg. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to prevent or treat vascular disease and / or an amount sufficient to inhibit angiogenesis.

The pharmaceutical composition of the present invention may be formulated into a unit dosage form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, suppositories, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

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 such as starch, calcium carbonate, sucrose, sucrose), 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 sterile aqueous solutions, non-aqueous solvents, 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 pharmaceutical composition of the present invention may be administered as an individual prophylactic agent or a therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

Further, the present invention provides a health functional food for preventing or ameliorating a vascular disease comprising the compound represented by the above-mentioned formula (1) or a salt thereof as an active ingredient.

In addition, the present invention provides a health functional food for prevention or improvement of vascular aging comprising the compound represented by the formula (1) or a salt thereof as an active ingredient.

The compound represented by the formula (1) or a salt thereof is not particularly limited as long as it can be usually prepared and / or purchased, but preferably it may be one which has been liberated from the mushroom.

The kind of health functional food is not particularly limited as long as it is usually produced and / or sold. For example, dairy products such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums and ice cream, soups, drinks, tea, drinks, alcoholic beverages, And can be used in the form of pills, powders, granules, infusions, tablets, capsules or beverages, all of which include health functional foods in the conventional sense.

The health beverage composition of the present invention is not particularly limited as long as it contains the compound represented by the above-mentioned formula (1) or a pharmaceutically acceptable salt thereof, but there is no particular limitation to the liquid ingredient, and various flavors or natural carbohydrates, ≪ / RTI > Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrins; And sugar alcohols such as xylitol, sorbitol, and erythritol. As natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have.

In addition to the above, the health functional food of the present invention may contain various kinds of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

In addition, the health functional foods of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination.

In addition, the present invention relates to a method for producing an extract, comprising the steps of: preparing an extract by mixing and extracting Cucurbitaceae mushroom and C ₁ -C ₄ alcohol; Fractionating the extract with an organic solvent to produce a fraction; And chromatographing the fractions to obtain a compound represented by the following formula (1): And a method for separating the compound represented by the formula (1).

The organic solvent is not particularly limited as long as it is usually used for separating a single component originating from a natural product. Preferably, the organic solvent may include at least one selected from the group consisting of nucleic acids, methylene chloride, ethyl acetate and C ₁ -C ₄ alcohol , More preferably methylene chloride and methanol.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

[ Example ]

Example  One. Mushroom mushroom  Extraction and fractionation of derived material

1-1. Spindle  Extract manufacture of mushroom

Three kilograms of dried mushroom (Pocheon mushroom), which was dried with the entire fruiting body, was pulverized to an appropriate size, 20 liters of 80% methanol was added, and extracted twice at room temperature for 24 hours , Filtered through a filter paper, and concentrated under reduced pressure to obtain 300 g of an extract.

1-2. Spindle  Organic solvent from mushroom extract Fraction  Produce

300 g of the extract prepared from Example 1-1 was suspended in 2 L of distilled water, 2 L of hexane was further suspended, and the suspension was divided into hexane, methylene chloride, ethyl acetate and butanol, , Ethyl acetate and butanol were evaporated and concentrated to give hexane, methylene chloride, ethyl acetate and butanol fractions.

1-3. Organic solvent From the fraction  activation Fraction  And preparation of compounds

The hexane, methylene chloride, ethyl acetate and butanol fractions obtained in Example 1-2 above were subjected to chromatography using a silica gel column to obtain another fraction. (V / v = 99%: 1%, 50%: 50%) and a methylene chloride-methanol mixed solvent (v / v = 98%: 2%, 50%: 50%) in a hexane-ethyl acetate mixed solvent (HEH1 to HEH6), 9 (HEM1 to HEM9), and 5 (HEE1 to HEE5, respectively) fractions of the herbaceous mushroom ethyl acetate fractions, ). Active fractions were obtained by confirming that HEH2, HEM2, HEM6, HEM7 and HEE3 in the fraction showed inhibitory activity in a concentration dependent manner on acetylcholinesterase.

1-4. activation From the fraction  Separation of single compounds

Among the active fractions of Examples 1-3, HEM2 was analyzed by HPLC as a fraction of Mulberry Mushroom Methylene Chloride, and Compound 1 was isolated and purified.

Comparative Example  One.

As the extract of Aspergillus oryzae, the compound determined by the following chemical structure was regarded as the present comparative example.

Comparative Example  2.

As the extract of Aspergillus oryzae, the compound determined by the following chemical structure was regarded as the present comparative example.

Comparative Example  3.

As the extract of Aspergillus oryzae, the compound determined by the following chemical structure was regarded as the present comparative example.

Comparative Example  4.

As the extract of Aspergillus oryzae, the compound determined by the following chemical structure was regarded as the present comparative example.

Comparative Example  5.

As the extract of Aspergillus oryzae, the compound determined by the following chemical structure was regarded as the present comparative example.

Experimental Example  1: Analysis of the structure of compound 1.

The Example 1-4 Compound 1 Molecular weight and molecular formula of separation in LC / MS spectrometer (manufacturer: Thermo LCQ deca finnigan plus) is determined by using a nuclear through the magnetic resonance (NMR) analyzer (Verian 500 MHz) ¹ 1 H NMR and ¹³ C NMR spectra were obtained to determine the molecular structure. ¹ H NMR results are shown in Fig. 1, ¹³ C NMR results are shown in Fig. 2, ESI-MS spectral analysis results are shown in Fig. 3, and IR spectral analysis results are shown in Fig.

As a result, the following chemical structure of Compound 1 was determined.

Compound 1:

1) Properties: White powder (melting point 182 ~ 184 ℃)

2) Molecular weight: 428 g / mol

3) Molecular formula: C ₂₈ H ₄₄ 0 ₃

^{4) 1 H NMR (500MHz,} CDCl 3), δ 6.51 (1H, d, J = 8.5Hz, H-7), 6.24 (1H, d, J = 8.5 Hz, H-6), 5.24 (1H, dd , J = 15.0, 8.0 Hz, H-23), 5.16 (1H, dd, J = 15.0, 8.0 Hz, H-22), 3.99 (1H, m, H-3), 1.03 (3H, d, J = 7.0 Hz, H-21), 0.94 (3H, d, J = 6.6 Hz, H-28), 0.88 (3H, s, H-19), 0.86 (3H, d, J = 6.4 Hz, H-26) , 0.83 (3H, s, H-18), 0.81 (3H, d, J = 6.4 Hz, H-27), ¹³ C NMR (125 MHz, CDCl ₃ ),? 135.6 (C-23), 132.9 (C-23), 130.9 (C-7), 82.3 (C-5), 79.6 ), 51.3 (C-9), 44.7 (C-13), 43.0 (C-24), 39.9 , 34.9 (C-1), 33.2 (C-25), 30.3 (C-2), 28.8 20.1 (C-26), 19.8 (C-27), 18.3 (C-19), 17.7 (C-28), 13.0 (C-18).

5) ESI-MS) data at m / z 451 [M + Na] + (calcd for C 28 H 44 0 3 Na:. 451)

6) Structure analysis technology for compounds

^The methyl groups (δH = 1.03, 0.94, 0.88, 0.86, 0.83, and 0.81) and the four methylene proton signals (δH = 6.51, 6.24, 5.24, and 5.16) Respectively.

* ¹³ C NMR spectra revealed 28 carbon signals and 7 methylene carbons (δC = 39.5, 37.2, 34.9, 30.3, 28.8, 23.6 and 20.8) and 5 methine carbons = 56.4, 51.9, 43.0, 39.9 and 33.2), 4 quaternary carbons (delta C = 82.3, 79.6, 44.7 and 37.1), 6 methyl carbons (delta C = 21.0, 20.1, 18.3, 17.7 and 13.0) and four olefinic carbons (? C = 135.6, 135.4, 132.5 and 130.9).

Preparation Example  1. Cell culture

(Dulbecco's modified Eagle's medium, Wuljin, Daegu, Korea) and 10% fetal bovine calf serum (Weljin, Daegu, Korea) Human fibroblasts were divided into 1 × 10 ⁵ cells. Then, the cells were cultured in a 5% carbon dioxide incubator at 37 ° C. for 3 days. When cells were cultured in 80-90% of the culture dish, the cells were treated with 1 ml of 2.5 × trypsin-ethylenediaminetetraacetic acid (EDTA) . Some of the isolated cells were maintained on subculture in calf fetal serum and DMEM, and some of them were used in the experiment.

The cells used for the experiment were washed twice with DMEM medium containing 1% penicillin-streptomycin solution (Weljin, Daegu, Korea) for removal of the cell culture medium for treatment with adriamycin, (Myondong Pharmaceutical, Seoul, Korea) for 4 hours.

The human fibroblast cells were cultured in a 100-mm diameter culture dish, except that umbilical vein cells purchased from Lonza were cultured with EGM-2 (Lonza, Walkersville, MD, USA) as an endothelial cell growth medium Cultured and harvested in the same manner as the culture, and treated with adriamycin.

The number of cell divisions was determined by measuring population doublings (PDs) while subculturing the cells. PD was obtained by using the formula of PD = log ₂ F / log ₂ I (F, final cell number), and human fibroblasts were PD <35 cells and cord blood cells were PD <30 cells Adriamycin was used for the treatment of old age cells, and cells with PD> 75 for human fibroblasts and PD> 50 for umbilical cord blood were used.

Experimental Example  2: Cytotoxicity measurement

In Preparative Example 1, adriamycin-treated cord blood cells were treated with 1 ml of 2.5 x trypsin-EDTA, and the cord blood cells were separated from the culture dish. Then, the separated cells were dispensed in a 50 ml tube of EGM-2 culture solution so that cells in the umbilical cord blood were 10,000 cells / ml, and 100 쨉 l were dispensed into each well of a 96-well culture container. The cells in the tube were divided into 1,000 cells.

Then, the cells were cultured in a 5% carbon dioxide incubator at 37 ° C for 24 hours, and 100 μl of EGM-2 culture was added to each well.

Then, 5 mM dimethyl sulfoxide (DMSO), 5 mM N-acetylcysteine, 500 mM Rapamycin, Compound 1 extracted in Example 1-4 or Comparative Examples 1 to 5 . 1 占 퐂 / ml of Compound 1 of Example 1-4 and Comparative Example 2 were added.

After incubation for 3 days in a 5% CO 2 incubator at 37 ° C, the degree of cell growth was confirmed by MTT assay. Specifically, 50 μl of 0.1% MTT solution (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl-2H-tetrazolium bromide) was added to each well of a 96- At 37 &lt; 0 &gt; C in a 5% CO2 incubator. Then, the culture solution and the MTT solution were removed, and 100 μl of dimethyl sulfoxide was added to dissolve the crystals. The absorbance was measured at 550 nm using a microplate reader. The measurement results are shown in Fig.

As shown in Fig. 5, it was confirmed that Compound 1 obtained in Example 1-4 did not show toxicity to cord blood and endothelial cells.

Experimental Example  3: Measurement of anti-aging activity of blood vessels

In Preparative Example 1, adriamycin-treated cord blood cells and fibroblasts were treated with 1 ml of 2.5 x trypsin-EDTA, and the cord blood cells or fibroblasts were separated from the culture dish. The cells were then divided into 12-well culture dishes or 24-well culture dishes.

Specifically, the fibroblasts obtained in Preparation Example 1 were added to a DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (Weljin, Daegu, Republic of Korea) in a 50 ml tube and in Preparation Example 1 for EGM- The cells and the culture medium were dispensed into each well of a 24-well culture container by placing the cells in the umbilical cord blood obtained. Finally, 5,000 fibroblasts per cell and 7,000 cells per umbilical cord were dispensed into a 12-well culture vessel. In a 24-well culture container, 3000 cells were injected per well and 5000 cells were injected into the umbilical cord blood.

The cells were cultured in a 5% CO 2 incubator at 37 ° C for one day. DMEM culture medium or EGM-2 culture medium containing 10% fetal bovine serum and 1% antibiotic was changed into each well, and the reagent was treated.

The above reagents were dissolved in dimethyl sulfoxide (DMSO), 5 mM N-acetylcysteine (NAC), 500 mM Rapamycin, Compound 1 extracted in Example 1-4 or Comparative Examples 1 - 5 was added. 0.1 μg / ml or 1 μg / ml of the compound of Compound 1 of Example 1-4 and Comparative Examples 1 to 5 was added.

After incubation for 3 days at 37 ° C in a 5% CO 2 incubator, the cells were washed twice with phosphate buffer, fixed with 3.7% paraformaldehyde for 1 min, and then fixed. The cells were then stained with SA-b-gal staining solution (40 mM citric acid / phosphate [pH 5.8], 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, _{mM MgCl 2, X-gal (} 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) 1 ㎎ / ㎖) the 12-well culture plate, the 1000 ㎕ or 24-well culture plate, the 500 ㎕ per well Was added.

Subsequently, the culture container to which the SA-b-gal staining solution was added was wrapped in silver foil and reacted at 37 ° C for 16 hours, washed twice with phosphate buffered saline (PBS) , St. Louis, MO, USA) for 1 minute. When crystals were formed during the dyeing, crystals were dissolved by DMSO. After the staining, the cells were washed twice with phosphate buffer, and then blue-stained cells were observed under an optical microscope to examine the effect on vascular cell senescence. The degree of SA-β-gal activity was expressed in percentage (%) by measuring the number of cells stained blue in the cytoplasm among 50-100 cells in total. Among the results of the optical microscope observation, the cells in the umbilical cord blood were shown in Fig. 6, the fibroblasts in Fig. 7, and the percentage of the measured cells in the cord blood was shown in Fig. 8 and the fibroblasts in Fig.

As can be seen in Figures 6 and 8, the cells treated with Compound 1 of Example 1 had significantly fewer blue stained cells than the cells treated with DMSO alone, and in particular, 1 μg of Compound 1 of Example 1 / Ml, the SA-b-Gal activity was reduced by about 40%, confirming the anti-aging activity similar to that of NAC and rapamvcin, which are conventionally known blood aging active substances.

Based on this, it can be seen that Compound 1 of the present invention can prevent or improve vascular diseases by inhibiting vascular endothelial cell senescence.

As can be seen in Figs. 7 and 9, it was confirmed that the compounds of Example 1 and Comparative Examples 1 to 5 do not have the activity inhibiting activity of SA-β-gal induced by adriamycin.

Thus, it was confirmed that the compound 1 of the present invention has anti-vascular endothelial cell senescence inhibitory activity, and fibroblasts, which are tissue-associated cells, do not exhibit anti-aging activity. Thus, Compound 1 of the present invention has a remarkable effect on inhibition of vascular cell senescence, and thus it can be effectively used for prevention or treatment of vascular diseases.

As described above, the compound 1 of the present invention has a high anti-aging activity and is highly likely to be utilized in various fields such as a pharmaceutical composition for preventing or treating vascular diseases, a pharmaceutical composition for inhibiting angiogenesis, and a health functional food Do.

[ Manufacturing example ]

Manufacturing example  1: Preparation of pharmaceutical composition

The preparation examples of the pharmaceutical composition comprising the compound 1 which is the herb spider mushroom-derived material of the present invention will be described, but the present invention is not intended to be limited thereto but will be specifically described.

Manufacturing example  1-1. Sanje  Produce

Compound 1 2 g

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

Manufacturing example  1-2. Manufacture of tablets

Compound 1 100 mg

Corn starch 100 mg

100 mg of milk

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

Manufacturing example  1-3. Preparation of capsules

Compound 1 100 mg

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

Manufacturing example  1-4. Manufacture of rings

Compound 1 1 g

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

Manufacturing example  1-5. Manufacture of granules

Compound 1 150 mg

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

Manufacturing example  2: Preparation of health functional foods.

Manufacturing example  2-1. Manufacture of Health Functional Foods

Compound 1 3000 mg

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

10 mg vitamin C

Biotin 10 μg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Potassium citrate 90 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is relatively mixed with a suitable preparation for health food, the compounding ratio may be arbitrarily changed, and the above ingredients may be mixed according to a conventional method for producing healthy food , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Manufacturing example  2-2. Manufacture of health drinks

Compound 1 3 g

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, Of health functional beverage composition.

Although the composition ratio is relatively mixed with the ingredients suitable for the favorite beverage as a preferable preparation example, the blending ratio may be arbitrarily varied depending on the regional and national preference such as the demand class, the demanding country, and the use purpose.

Claims (10)

delete delete delete A pharmaceutical composition for preventing or treating dementia caused by vascular endothelial cell senescence, which comprises a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient and has a function of inhibiting SA-? -Galactosidase activity Or &lt; / RTI &gt;
[Chemical Formula 1]

The pharmaceutical composition for preventing or treating dementia according to claim 4, wherein the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof is derived from a spore mushroom.
delete delete A pharmaceutical composition for preventing or treating dementia caused by vascular endothelial cell senescence, which comprises a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient and has a function of inhibiting SA-? -Galactosidase activity Or health functional food for improvement.
[Chemical Formula 1]

delete delete

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