KR101528190B1 - Composition for inhibiting cellular senescence comprising 2,5-dihydroxy-4-methoxyacetophenone isolated from Paeonia suffruticosa - Google Patents

Abstract

The present invention relates to a composition for inhibiting cell senescence comprising 2,5-dihydroxy-4-methoxyacetophenone as an active ingredient, which is isolated from Mulberry, And a pharmaceutically acceptable carrier. The present invention also provides a pharmaceutical composition for inhibiting cell senescence. By inhibiting the cell aging process of the fibroblast, it can be useful for treating diseases such as aging-related diseases such as skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damaged tissue, arteriosclerosis, prostatic hyperplasia and liver cancer . It is also expected to be useful for development of anti-aging functional food, anti-aging drug, and cosmetics which can inhibit cell senescence.

Description

A composition for inhibiting cell senescence comprising 2,5-dihydroxy-4-methoxyacetophenone as an active ingredient, which comprises a 2,5-dihydroxy-4-methoxyacetophenone,

The present invention relates to a composition for inhibiting cell senescence comprising 2,5-dihydroxy-4-methoxyacetophenone (2,5-dihydroxy-4-methoxyacetophenone)

When normal somatic cells are divided a certain number of times, they can no longer divide and become aged. This is because the telomere at the end of the chromosome is becoming shorter and shorter in the process of cell division, resulting in DNA damage. Cellular senescence is induced not only by shortening of telomere but also by dysfunction of cancer gene and cancer suppressor gene, inflammation reaction, oxidative stress, anticancer agent, ultraviolet ray and radiation. Aging cells are large in size, flatter in shape, stop cell growth, have many signs of DNA damage in the nucleus, and secrete a variety of inflammatory proteins. And biochemically it is known that senescence-associated β-galactosidase (SA-β-gal) activity increases. Although senescence is induced by a variety of factors, it has been shown that senescence is regulated through the signaling pathway of p53 and Rb / p16 tumor suppressor genes.

Cell senescence also inhibits or promotes cancer, and is suggested as an important mechanism of tissue regeneration and restoration, tissue / individual aging and aging related diseases. In addition, cellular senescence contributes to the pathogenesis of various aging-related diseases such as cancer, arteriosclerosis, skin aging, degenerative neurological diseases, myopenia, osteodystrophy, and hyperplasia of the prostate. Recent studies have shown that selective regulation of cell senescence can regulate tissue, organ aging, health life span, and the development of aging-related diseases. Telomerase-deficient mice are known to be aging rapidly, confirming that increasing telomerase expression in old telomeres-deficient mice reverses the degenerative changes of tissues or organs due to aging. In a mouse model with rapid senescence, selective removal of cells expressing p16, which is known to increase expression in senescent cells, inhibits senescence-induced tissue lesions and reduces the incidence of aging-related diseases. In the course of hepatic fibrosis in mice, aging of hepatic stellate cells occurs. It is known that aging of hepatic stellate cells inhibits excessive liver fibrosis. If the p53 activity is not properly regulated, the aging will occur quickly, but the proper p53 activity is known to inhibit aging.

Studies have also been reported on substances that have the potential to inhibit cellular senescence. Drugs or single components such as vitamin C, N-acetylcysteine, NS398 and epifriedelanol inhibit this cell senescence. In rapamycin-induced mouse models, 4,4'-diaminodiphenylsulfone has been reported to inhibit the development of aging-related diseases in small nematodes and to increase health life span.

The rootstock is a root shell of Paeonia suffruticosa Andrews belonging to Paeoniaceae. It has been widely used because it has hypercholesterolemia, hypercholesterolemia, reperfusion ischemia. It has been reported that the extracts of Murine extract inhibit the release of IL-8 and MCP-1, which are chemokine in vivo, and also inhibit the expression of iNOS and COX-2, inflammatory mediators. It is known that the extract of Mulberry extract has excellent inhibitory activity against HIV integrase, and paeonol, which is a main component of the herringbone, suppresses the production of active oxygen by inhibiting xanthine oxidase, It is known to inhibit the release of TNF-α, an inflammatory mediator, but its efficacy in controlling cellular senescence is not yet known.

Korean Patent No. 10-0757219 discloses an antimicrobial composition comprising a herringbone extract or compounds isolated therefrom. More particularly, the present invention relates to an antimicrobial composition comprising a extract of Moutan Cortex Radicis, a root shell of Paeonia suffruticosa, The compounds isolated from the present invention exhibit inhibitory activity against the common infectious disease strain and thus can be used as an antimicrobial composition. However, in the present invention, 2,5-dihydroxy-4-methoxyacetophenone 2,5-dihydroxy-4-methoxyacetophenone) inhibited fibroblastogenesis.

An object of the present invention is to provide a composition for inhibiting cell senescence containing 2,5-dihydroxy-4-methoxyacetophenone (2,5-dihydroxy-4-methoxyacetophenone) have.

In order to achieve the above object, the present inventors investigated the cell aging inhibitory effect of eight single components (MM1-8) isolated from Korean herbal fibroblasts. The cell aging inhibition efficacy was confirmed by SA-β-gal active staining, p53 expression analysis and active oxygen analysis. As a result, it was confirmed that a single ingredient MM-2 (2,5-dihydroxy-4-methoxyacetophenone) in human fibroblasts is effective for inhibiting cell senescence and completed the present invention.

The present invention provides a pharmaceutical composition for inhibiting cell senescence comprising 2,5-dihydroxy-4-methoxyacetophenone represented by the following formula (1) as an active ingredient. Specifically, the cell is a fibroblast.

≪ Formula 1 >

R ₁ = OH, R ₂ = OCH ₃ , R ₃ = OH, and R ₄ = CH ₃ .

More particularly, the 2,5-dihydroxy-4-methoxyacetophenone is isolated from the extract of Miltini, and more particularly, (CH ₂ Cl ₂ ) fraction extract obtained by adding distilled water and dichloromethane (CH ₂ Cl ₂ ) and fractionating the extract.

The 2,5-dihydroxy-4-methoxyacetophenone of the above formula (1) can be isolated from a natural substance, preferably a plant. It can be extracted by extracting various organ, root, stem, leaf, flower as well as plant tissue culture of natural, hybrid, and variant plants. Most preferably, it can be separated from the sheath.

Specifically, the cell senescence is characterized by being induced by adriamycin, and inhibition of cell senescence is measured by measuring senescence-associated beta-galactosidase (SA-beta-gal) .

In the case of the pharmaceutical composition of the present invention, the pharmaceutical composition may contain a pharmaceutically acceptable carrier besides 2,5-dihydroxy-4-methoxyacetophenone Such pharmaceutically acceptable carriers are those conventionally used in pharmaceutical preparations and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, But are not limited to, cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like . In addition, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. as an additive.

In the above pharmaceutical composition, the administration method is determined according to the degree of symptom of cell senescence. Usually, local administration method is preferable. The dosage of the active ingredient in the pharmaceutical composition may vary depending on the route of administration, the severity of the disease, the age, sex, and weight of the patient, and may be administered once to several times per day.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The pharmaceutical composition may be prepared in unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient, or may be prepared by inserting it into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions, or may be in the form of elixirs, excipients, powders, granules, tablets, alerts, lotions, ointments and the like.

Meanwhile, the pharmaceutical composition may treat any one selected from the group consisting of skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin injured tissue, arteriosclerosis, prostatic hyperplasia and liver cancer, but is not limited thereto.

The inventors of the present invention found that 2,5-dihydroxy-4-methoxyacetophenone (2,5-dihydroxy-4-methoxyacetophenone) isolated from Momomori suppresses cell aging by adriamycin, Respectively. By inhibiting the cell senescence process, it can be useful for treating diseases such as aging-related diseases such as skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damaged tissue, arteriosclerosis, prostatic hyperplasia and liver cancer. It is also expected to be useful for the development of anti-aging functional food, anti-vascular aging drug, and cosmetics that can inhibit cellular senescence of fibroblasts.

Fig. 1 is a schematic view showing the separation of the herringbone extract.
Fig. 2 shows the chemical structure of a single component separated from the dorsal root.
FIG. 3 shows the cytotoxicity of a single component of the herringbone in human fibroblasts and the effect of inhibiting cell senescence by adriamycin. A, Cytotoxic effect of single component of vermicelli. Each component was treated with 10 ug / ml, cultured for 3 days, and cytotoxic effect was investigated by MTT method. B, SA-β-gal active stain percentage. After treatment with adriamycin, MM-3 and MM-5 were treated with 10 ug / ml, and SA-β-gal active staining was performed after 3 days. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine. * p < 0.05 vs. DMSO.
4 shows the cytotoxicity of MM-2 [2,5-dihydroxy-4-methoxyacetophenone] in human fibroblasts and the cytotoxic effect of adriamycin on cell senescence . A, and MM-2. MM-2 was treated with 0.1, 0.1 ug / ml for 3 days, and cytotoxic effect was investigated by MTT method. B, SA-β-gal active staining photograph. After treatment with adriamycin, MM-2 was treated with 0.1 μg / ml and SA-β-gal active staining was performed after 3 days. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. C, p53, phosphorylated S6K, p21. After treatment with adriamycin, MM-2 was treated with 0.1 ug / ml and the degree of expression of each protein was examined by Western blotting. NT, untreated; Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin.
Figure 5 shows the effect of MM-2 [2,5-dihydroxy-4-methoxyacetophenone] on the production of reactive oxygen species in human fibroblasts. A, active oxygen flow cytometry. B, the average amount of active oxygen. The fibroblasts were treated with adriamycin, treated with MM-2 at 0.1 ug / ml, and the level of active oxygen in the cells was examined by flow cytometry. Each experiment was repeated three times or more independently and then expressed as mean and standard deviation. ADR, adriamycin; Y, young cells; Cont, control group; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin.
Fig. 6 shows the effect of MM-2 [2,5-dihydroxy-4-methoxyacetophenone] on human cell fibroblasts in the inhibition of cell senescence. A, SA-beta-gal active staining and SA-beta-gal active staining percentage. B, and MM-2. Reproductive aging cells were treated with 0.1 μg / ml of MM-2 and subjected to SA-β-gal active staining three days later. Cytotoxicity was assessed by MTT assay. The results were expressed as mean and standard deviation after each experiment repeated 3 times or more independently. Old, Old cells; DMSO, dimethylsulfoxide; NAC, N-acetylcysteine; Rap, rapamycin. * p < 0.05 or ** p < 0.01 vs. DMSO.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< Example  1> Root Extract Separation and Chemical Composition of Single Components

1. Root extract and single component separation

9.6 kg of the herringbone was extracted twice with 70% MeOH (10 L × 2) while refluxing at 60 ° C. for 12 hours and concentrated under reduced pressure to obtain 2.3 kg of MeOH extract. To this MeOH extract was added 1.0 L of H ₂ O and shaken to give a suspension. The same amount of CH ₂ Cl ₂ , EtOAC was added and the solvent fraction was diluted with CH ₂ Cl ₂ 80.3 g of fraction, 181.6 g of EtOAC fraction and 1109.32 g of water layer were obtained.

A silica-gel (230-400 mesh) column with a size of 70 × 6.5 cm was prepared, and 80.3 g of CH ₂ Cl ₂ fraction was loaded. The mobile phase was eluted with a gradient elution [CH ₂ Cl ₂ : MeOH: H ₂ O], and 16 subfractions (MMF 1-16) were obtained by TLC. (36.4 g) and MM-5 (4.9 g) were obtained by recrystallization of fraction MMF3 (51.1 g) and fraction MMF11 (6.1 g). MMF-2 (19.9 mg) was prepared by loading the fractions MMF5 (600.0 mg) and fraction MMF10 (800.0 mg) onto a 75 × 2.0 cm RP-18 column and eluting with mobile phase solvent [MeOH: H ₂ O] MM-4 (24.8 mg) and MM-8 (11.5 mg). (11.5 mg) and MM-6 (10 mg) were prepared by loading fraction MMF7 (2.8 g) and fraction MMF13 onto a 75 × 4.0 cm RP-18 column and eluting with mobile phase solvent [MeOH: H ₂ O] 630.0 mg) and MM-7 (594.0 mg) were obtained (Fig. 1). The chemical structure of the compounds was determined by using spectroscopic methods such as MS, NMR ( ¹ H, ¹³ C, DEPT, ¹ H- ¹ H COZY, HMQC and HMBC) Respectively. Each fraction was dissolved in dimethyl sulfoxide and treated with cells.

2. Physico-chemical properties of separated materials

The eight compounds isolated from the CH ₂ Cl ₂ fraction in the woody part were analyzed by various spectroscopic methods such as MS and NMR ( ¹ H, ¹³ C, DEPT, ¹ H- ¹ H COZY, HMQC, and HMBC) MM-1 [paeonol], MM-2 [2,5-dihydroxy-4-methoxyacetophenone], MM-3 [ MM-4 [benzoic acid], MM-5 [benzoylpaeoniflorin], MM-6 [paeonoside], MM-7 [ Paeoniflorin], and MM-8 [oxypaeoniflorin] (Fig. 2).

MM -1 (paeonol) : white crystals; ^{1 H-NMR (250MHz, CDCl} 3): δ2.57 (3H, s), 3.85 (3H, s), 6.32 (1H, d, J = 2.5Hz, 3H), 6.36 (1H, dd, J = 2.5, 8.7 Hz, 5-H), 7.65 (1H, d, J = 8.7 Hz, 6-H), 12.7 (1H, s, COOH).

^{13 C-NMR (63MHz, CDCl} 3): δ26.2 (-COCH 3), 55.5 (-OCH 3), 100.8 (C-3), 107.6 (C-5), 113.9 (C-1), 132.3 ( C-6), 165.2 (C -2), 166.1 (C-4), 202.6 (-COCH 3)

MM -2 (2,5-dihydroxy-4 -methoxyacetophenone): 1 H-NMR (250MHz, CD 3 OD): δ2.36 (3H, s), 3.76 (3H, s), 6.30 (1H, s), 7.0 (1 H, s).

^{13 C-NMR (63MHz, CD} 3 OD): δ26.5 (-COCH 3), 56.3 (-OCH 3), 100.7 (C-5), 116.0 (C-6), 113.3 (C-1), 140.1 (C-3), 157.0 ( C-2), 159.7 (C-4), 204.2 (-COCH 3).

MM -3 (acetovanillone) : white power; ¹ H-NMR (250 MHz, CD ₃ OD):? 2.52 (3H, s), 3.93 (3H, s), 7.00 (1H, d, J = 8.4,6- = 2.12, 3-H), 7.54 (1H, dd, J = 8.44, 2.12, 5-H).

^{13 C-NMR (63MHz, CD} 3 OD): δ26.3 (-COCH 3), 56.4 (-OCH 3), 111.7 (C-6), 115.7 (C-3), 123.1 (C-5), 131.7 (C-1), 147.6 ( C-2), 153.8 (C-4), 199.6 (-COCH 3)

MM -4 (benzoic acid) : colorless amorphous powder; ¹ H-NMR (250 MHz, CDCl ₃ ):? 8.14 (2H, d, J = 7.2,2,6-H), 7.40-7.64 (3H, m, , br, s, COOH)

¹³ C-NMR (63 MHz, CDCl ₃ ):? 129.29 (C-1), 128.43 (C-3, 5), 130.17 )

MM -5 (benzoylpaeonifluorin) : colorless amorphous powder; ¹ H-NMR (250 MHz, CD ₃ OD):? 1.13 (3H, s), 5.28 (1H, s, 9-H, acetal proton), 7.34-7.94 (10H, benzoyl-H).

^{13 C-NMR (63MHz, CD} 3 OD): δ19.5 (C-10), 23.0 (C-7), 43.7 (C-5), 44.4 (C-3), 61.6 (C-8), 65.2 (C-6 '), 71.9 (C-6), 74.9 (C-2'), 75.1 C-5 ''), 129.7 (C-3 '', C-5 ''), 100.0 (C-1 ' ) 130.1 (C-2 "', C-6"'), 130.6 (C-2 " 4 "), 134.5 (C-4"), 167.6 (C-7 "), 167.9 (C-7"').

MM -6 (paeonoside) : white power; ^{1 H-NMR (250MHz, CD} 3 OD): δ2.50 (3H, s, COCH 3), δ3.70 (3H, s, OMe), 4.92 (1H, d, J = 7.2, 1'-H) , 3.16-3.80 (6H, glucose-H), 6.70 (1H, d, J = 2.3Hz, 3-H), 6.36 (1H, dd, J = 2.3, 9.0Hz, , d, J = 9.0 Hz, 6-H),

^{13 C-NMR (63MHz, CD} 3 OD): δ32.15 (-COCH 3), 56.17 (-OCH 3), 62.55 (C-6 '), 71.33 (C-4'), 74.85 (C-2 ' ), 78.32 (C-5 '), 78.51 (C-3'), 102.50 (C-1 ', C-3), 109.44 , 166.29 (C-4), 200.53 (-COCH 3).

MM- 7 (paeoniflorin) : white powder; ¹ H-NMR (250 MHz, CD ₃ OD):? 1.26 (3H, s, 10-H), 1.72 (1H, d, J = 12.5 Hz) (1H, d, J = 11Hz), 2.41 (1H, dd, J = 7.2,11Hz) H), 7.34-7.96 (5H, benzoyl-H), 3.14-3.78 (6H, glucose-H)

^{13 C-NMR (63MHz, CD} 3 OD): δ19.6 (C-10), 23.3 (C-7), 43.8 (C-5), 44.4 (C-3), 61.6 (C-8), 62.8 (C-6 '), 71.6 (C-4'), 72.1 (C-6), 74.9 2), 89.0 (C-1), 100.1 (C-1 '), 102.2 (C-9), 106.3 ", C-6"), 131.1 (C-1 "), 134.4 (C-4"), 167.9 (C-7 ").

MM- 8 (oxypaeonifluorin) : white power; ^{1 H-NMR (250MHz, CD} 3 OD): δ1.23 (3H, s, 10-H), 1.67 (1H, d, J = 11Hz), 2.35 (1H, dd, J = 6.8, 11Hz) (6 (1H, d, J = 10.6 Hz), 2.06 (1H, d, J = 12.5 Hz) (2H, d, J = 8.8 Hz), 7.70 (2H, d, J = 8.8 Hz).

^{13 C-NMR (63MHz, CD} 3 OD): δ19.3 (C-10), 23.1 (C-7), 43.6 (C-5), 44.2 (C-3), 60.9 (C-8), 62.5 (C-6 '), 71.4 (C-4'), 72.0 (C-6), 74.7 2), 89.0 (C-1), 99.9 (C-1 '), 102.0 (C-9), 106.1 ", C-6"), 121.7 (C-1 "), 163.4 (C-4"), 167.7 (C-7 ").

< Example  2> Cytotoxicity and cell aging inhibitory effect of single component of vermicelli

1. Experimental material

Human fibroblasts and umbilical vein endothelial cells were purchased from Lonza (Walkersville, MD, USA). (Penicillin-Streptomycin, WelGene (Daegu, Korea), Endothelial Cell Growth Medium-2 (DMEM), Dulbecco's Modified Eagle's Medium -2, EGM-2) were purchased from Lonza (Walkersville, MD, USA). Antibodies to p53 were obtained from Santa Cruz Biotech, Inc. (SantaCruz, CA, USA), and antibodies against p21 and pS6 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). The GAPDH antibody was obtained from Dr. Kwon Sun - sun of Korea Biotechnology Research Institute. Adriamycin was manufactured by Ildong Pharmaceutical Co., Ltd.

2. Cell culture

Human fibroblasts were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotic (penicillin 10,000 цm / ml, streomycin 10,000 ug / ml) 10 ⁵ , and then cultured in a 5% carbon dioxide incubator at 37 ° C. If the cells were grown at the bottom of the culture dish by 80-90%, trypsin-EDTA solution (2.5X) was added to separate the cells, followed by subculture. Cells in umbilical cord blood were cultured in the same manner as human fibroblasts using EGM-2 as a culture medium. The number of cells was measured every time the cells were transferred, and the number of times of division of the cells was examined. The population doubling (PD) was calculated using the equation: PD = log ₂ F / log ₂ I (F = final cell number, I = initial cell number). The cells used for the experiment were PD <35 or PD> 75 in the case of human fibroblasts, and PD <30 or PD> 50 in the cord blood.

3. Induction of cell aging by treatment with adriamycin

Human fibroblasts and umbilical vein endothelial cells were plated at a density of 1.5x10 ⁵ cells in a 100 mm diameter culture dish. After culturing for 3 days at 37 ° C in a 5% carbon dioxide incubator, the cell culture medium was removed. Cells were washed twice with DMEM containing antibiotics. Cells were treated with 500 nM adriamycin for 4 hours and then washed three times with DMEM medium containing antibiotics. Human fibroblasts were cultured in DMEM containing 10% fetal bovine serum and 1% antibiotic, and human umbilical vein endothelial cells were cultured in EGM-2. After 4 days, it was confirmed that senescence-associated β-galactosidase (SA-β-gal) staining resulted in cell senescence.

4. Measurement of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT)

The effect of single compound on the cell growth rate was investigated by MTT method. 0.1% MTT solution was added to each well of a 96-well culture vessel in an amount of 50 μl per well and reacted in a 5% carbon dioxide incubator at 37 ° C for 3 hours. After removing the culture solution and MTT solution, 100 μl of dimethyl sulfoxide was added to dissolve the crystals formed. The cell growth rate was measured by measuring the absorbance at 550 nm using a microplate reader.

5. Investigation of the effect of single component of the herringbone on the cell aging by adriamycin

We examined the effect of single compounds isolated from Mulberry on aged cells by adriamycin. Cells treated with adriamycin for 4 hours were separated from the culture dish with trypsin-EDTA and dispensed into 96-well, 12-well, 24-well cell culture vessels. In a 96-well cell culture container, 500 fibroblasts and 1,000 cells were placed in each well. 24 wells were plated at 3000 cells / well, cord blood cells were plated at 5000 cells / well and 12 wells were plated at 5000 cells / well and 7000 cells / well of vascular endothelial cells. And cultured in a 5% carbon dioxide incubator at 37 ° C for one day. In 96 wells, 100 μl of DMEM culture medium containing 10% fetal bovine serum and 1% antibiotic and 100 μl of EGM-2 culture medium were added to each well. After 12 and 24-well cultures were exchanged, &lt; / RTI &gt; ug / ml. Dimethylsulfoxide was used as a negative control, and 5 mM N-acetylcysteine and 500 nM rapamycin were added as a positive control. After 3 days of incubation at 37 ° C in a 5% carbon dioxide incubator, the degree of cell growth was measured by MTT method and the degree of cell senescence was measured by senescence-associated β-galactosidase (SA-β-gal) Staining method.

6. senescence-associated β-galactosidase (SA-β-gal) active staining

The effect on cell senescence was examined by SA-β-gal active staining. After 3 days of treatment with a single compound in a 24 well or 12 well culture vessel, the cells were washed with phosphate buffer. The cells were fixed with 3.7% paraformaldehyde, and the fixative solution was removed and washed again with phosphate buffer solution. SA-β-gal staining solution [40 mM citric acid / phosphate; pH 5.8, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl ₂ , X-gal 1 mg / ml] was added to each well 250 ul per well and 500 ul per well in a 12 well culture vessel. Wrapped in silver foil and reacted at 37 캜 for 17 hours. After washing twice with phosphate buffered saline (PBS), the cells were stained with 1% ezine solution for 1 minute. After washing twice with phosphate buffer, blue-stained cells were observed under an optical microscope. 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.

7. Cell protein extraction

Each cell was placed in a 60 mm culture dish at 1 × 10 ⁵ cells and cultured at 37 ° C. in a 5% CO 2 incubator. Cells were washed twice with DMEM medium containing antibiotics, then treated with a single component of the herringbone for 1 hour before concentration and treated with 500 nM of adriamycin for 4 hours. After the culture medium was removed, the cells were washed twice with phosphate buffer. The cell lysate solution (25 mM Tris-HCl, pH 7.6), 150 mM Nacl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium vanadate vanadate), 5 mM NaF, protease inhibitor or 1 mM PMSF]. The cells were scraped using a cell scraper to scrape the culture dish, and then transferred to a microneedle tube. The solution was shaken every 10 minutes while reacting on ice for 30 minutes. The supernatant was transferred to a new tube by spinning at 12,000 rpm for 15 minutes. The amount of protein in the solution was quantified by bicinchoninic acid (BCA) method (Pierce Biotechnology Inc., Rockford IL, USA) using bovine serum albumin as a standard protein.

8. Western blot analysis

Protein (30 μg) was separated by electrophoresis on a 10% SDS-polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and allowed to react for 30 minutes in Tween-20-Tris buffered saline (TTBS) containing 5% whole milk powder. Nitrocellulose membranes were reacted overnight in 5% whole milk powder-TTBS solution containing primary antibody against p53 or p21. After washing three times for 10 minutes with TTBS solution, the cells were reacted with a secondary antibody conjugated with horseradish peroxidase for 1 hour and 30 minutes. The membranes were washed five times for 7 min with TTBS and the amount of p53 or p21 was measured using an enhanced chemiluminescence solution. The amount of specific protein reacted with each antibody was measured using a LAS-3000 imaging device (Fujifilm Corp., Stanford, CT, USA). The same amount of protein used in each experiment was compared using glyceraldehyde-3-phosphate dehydrogease (GAPDH) antibody.

9. Measurement of intracellular reactive oxygen (ROS) concentration

Cells were plated at ^1.5 × 10 ⁵ in a 100 mm culture dish and cultured in a 5% carbon dioxide incubator at 37 ° C. for 3 days. Cells were washed twice with DMEM medium containing antibiotics and treated with 500 nM adriamycin for 4 hours. The cells were washed once with phosphate buffer and treated with trypsin-EDTA solution (2.5%), and then the cells were divided into 1 × 10 ⁵ cells in a 60 mm culture dish. And cultured in a 5% carbon dioxide incubator at 37 ° C for one day. The culture medium was changed and a single compound was treated at each concentration. As a negative control, dimethyl sulfoxide was added as a positive control, 5 mM N-acetylcysteine and 500 nM rapamycin. After incubation for 3 days in a 5% CO 2 incubator at 37 ° C, the cells were washed twice with DMEM containing antibiotics and treated with 250 μM H ₂ DCFDA for 20 minutes. The cells were washed once with phosphate buffered saline, and the trypsin-EDTA solution was added to the cells. The supernatant was discarded at 5,000 × g for 1 minute, and the cells were washed with 1 ml of phosphate buffer solution containing 2% fetal bovine serum, and further washed at 5,000 × g for 1 minute. The washing procedure was repeated twice, followed by the addition of 1 ml of 1% paraformaldehyde. The amount of intracellular ROS was measured using a BD FACS Canto II flow cytometer (BD Biosciences, San Jose, Calif.).

10. Results

We investigated which compounds in the fibroblasts inhibited cell senescence by adriamycin. First, each single component was treated with 10 ug / ml to observe cytotoxicity. No cytotoxicity was seen in the single component except MM-2 (Fig. 3A). Among them, MM-3 and MM-5 were examined for their ability to inhibit cell senescence by adriamycin. It was confirmed that MM-3 and MM-5 did not inhibit SA-β-gal activity increased by adriamycin and thus did not inhibit cell aging (FIG. 3B).

When the concentration of MM-2 was treated with 1, 0.1 ug / ml to examine cytotoxicity, no cytotoxicity was observed at 0.1 ug / ml (FIG. 4A). MM-2 inhibited adriamycin-induced cell senescence by SA-β-gal staining and analysis of p53 and p21 protein expression. MM-2 inhibited increased SA-β-gal activity by adriamycin (FIG. 4B) but did not decrease p53, p21 protein expression (FIG. 4C). As a result of investigating whether MM-2 reduces the amount of intracellular ROS that is increased by adriamycin, the amount of ROS did not change even after treatment with MM-2 (FIG. 5). We investigated whether MM-2 inhibits adipocyte-mediated cell senescence in response to adriamycin-induced cell death. When MM-2 was treated with replicating senescent cells, SA-b-gal activity staining decreased (Fig. 6A), but did not show cytotoxicity (Fig. 6B).

These results suggest that MM-2 (2,5-dihydroxy-4-methoxyacetophenone) inhibits cell senescence in fibroblasts.

Claims (7)

(2,5-dihydroxy-4-methoxyacetophenone) represented by the following formula (1) as an active ingredient and inhibiting cell senescence Or a pharmaceutically acceptable salt thereof.
&Lt; Formula 1 >

R ₁ = OH, R ₂ = OCH ₃ , R ₃ = OH, and R ₄ = CH ₃ . delete The method according to claim 1, wherein the herringbone extract is a dichloromethane (CH ₂ Cl ₂ ) fraction extract obtained by extracting and fractionating distilled water and dichloromethane (CH ₂ Cl ₂ ) Or &lt; / RTI &gt; The pharmaceutical composition for preventing or treating skin aging according to claim 1, wherein the cell is a fibroblast. The pharmaceutical composition for preventing or treating skin aging according to claim 1, wherein the cell aging is induced by adriamycin. The pharmaceutical composition for preventing or treating skin aging according to claim 1, wherein the inhibition of cell senescence measures the inhibition of senescence-associated beta-galactosidase (SA-beta-gal) activity. delete

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