KR20140032201A - Composition for inhibiting cellular senescence comprising loliolide - Google Patents

Abstract

The present invention relates to a composition for inhibiting cellular senescence which includes (-)-loliolide as an active ingredient. The present invention provides the composition for inhibiting cellular senescence which inhibits senescence of fibroblast or replicative senescence which is induced by adriamycin. More specifically, the (-)-loliolide is isolated from Polygoniavicularisherba extract. By inhibiting cellular senescence of human fibroblast, the composition for inhibiting cellular senescence which includes (-)-loliolide as an active ingredient can be used for senescence related diseases such as aging of the skin, rheumatoid arthritis, degenerative arthritis, hepatitis, chronic skin tissue damage, hardening of the artery, benign prostatic hyperplasia, liver cancer, and the likes.

Description

[0001] The present invention relates to a composition for inhibiting cellular senescence comprising laurylide as an active ingredient,

The present invention relates to a composition for inhibiting cell senescence comprising a lauride [(-) - loliolide] as an active ingredient.

Normal somatic cells divide into a certain number of times and can no longer divide, resulting in cell aging. This is due to the fact that telomeres at the chromosome end become shorter and shorter in the process of cell division, resulting in DNA damage, which is called reproductive aging (Cell. (2007) 130: 223-233). Cellular senescence is induced not only by shortening of telomeres but also by dysfunction of cancer gene and cancer suppressor gene, inflammation reaction, oxidative stress, anticancer agent, ultraviolet ray and radiation (Genes & Development. (2010) 24: 2463-2479). 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 (J Cell Biol. (2011) 192: 547-556). It is known that senescence-associated β-galactosidase (SA-β-gal) activity increases biochemically (Proc Natl Acad Sci U S A. (1995) 92: 9363-9367). Although senescence is induced by a variety of factors, aging has been shown to be regulated through the signaling pathway of p53 and Rb / p16 tumor suppressor genes (Curr Opin Genet Dev. (2011) 21: 107-112).

Cell aging may also inhibit or promote cancer, and has been suggested as an important mechanism of tissue regeneration and repair, tissue / individual aging and aging-related diseases. Cell senescence also contributes to the pathogenesis of various aging-related diseases such as cancer, arteriosclerosis, skin aging, degenerative neurological diseases, myopenia, osteodystrophy, and prostatic hypertrophy. Recent studies have reported that selective control of cell aging can control the development of tissues, organs, aging, health life, and age-related diseases. Telomerase deficient mice are known to be aging rapidly, and it has been shown that increasing telomerase expression in old telomere-deficient mice reverses the degenerative changes of tissues or organs due to aging (Nature. (2011) 469: 102 -106). The selective removal of p16-expressing cells, which are known to increase expression in senescent cells in a rat model with rapid aging, has been shown to inhibit senescence-induced tissue lesions and reduce the incidence of aging-related diseases (Nature 2011) 479: 232-236). In the process of liver fibrosis in mice, aging of hepatic stellate cells appears, and aging of hepatic stellate cells is known to function to inhibit excessive liver fibrosis. If p53 activity is too high in an unregulated state, aging occurs quickly, but proper p53 activity is known to inhibit aging.

In addition, the results of research on substances that are effective in inhibiting cell aging have been reported. Drugs or single components such as vitamin C, N-acetylcysteine, NS398 and epifriedelanol inhibit this cell senescence (Nutrition Research and Practice. (2007) 1: 105-112; Mech Ageing Dev. (2008) 129: 706-713, Planta Med. (2011) 77: 441-449). And rapamycin has been reported to inhibit the development of aging-related diseases in small nematodes and increase health life span in mice model of 4,4'-diaminodiphenylsulfone ( Nature. (2009) 460: 392-395; Proc Natl Acad Sci US A. (2010) 107: 19326-19331).

In the mouse model, the sperm motility is increased by exposure to electromagnetic waves, the gingival inflammation is relieved in humans, the liver fibrosis caused by the bile duct ligation is inhibited, and acetaminophen is inhibited. (2011) 14: 720-724; Environ Toxicol Pharmacol. (2009) 27 (2009) 27 It has been known that there are various effects such as relaxation of vascular smooth muscle cells and expansion of blood vessels. : 225-230; J Ethnopharmacol. (2005) 99: 113-117).

On the other hand, in Korean Patent Laid-Open No. 10-2011-0041710, the inventors of the present invention have disclosed a method for producing rhizomes of Rhei rhizoma, Cirsii Radix, Plantaginis semen, Cinnamoni cortex, Cinnamoni cortex spissus, A pharmacological agent for inhibiting senescence comprising, as an active ingredient, one or two herbal medicine extracts selected from the group consisting of Euonimi lignum suberalatu, Salicis radicis cortex, Polygoni avicularis herba, and Chaenomelis langenariae radix Although the composition is disclosed, there is no mention of the compound of the present invention [(-) - loliolide].

It is an object of the present invention to provide a composition for inhibiting cell senescence comprising a lauride [(-) - loliolide] as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition containing a lauride [(-) - loliolide] which can exhibit a therapeutic effect such as skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damaged tissue, arteriosclerosis, prostatic hyperplasia, And to provide a pharmaceutical composition for inhibiting cell senescence.

In order to achieve the above object, the present invention was to investigate the cytotoxic effect of twelve single components isolated from human fibroblasts and umbilical vein endothelial cells. Among them, it has been confirmed that the laurylide [(-) - loliolide] inhibits cell senescence by adriamycin in human fibroblasts, and inhibits cell senescence even in cells induced by replication senescence.

A composition for inhibiting cell senescence comprising a lauride [(-) - loliolide] represented by the following formula (1) as an active ingredient. Specifically, the Lowry O fluoride [(-) - loliolide] is Single shaft (Polygoni characterized in that separate from the avicularis herba) extract, more specifically the Single shaft extract Single shaft (Polygoni avicularis herba) is added to distilled water and hexane (n-hexane), and ethyl acetate (EtOAc) is added to the distilled water layer for fractionation.

≪ Formula 1 >

The lauride [(-) - loliolide] of formula (1) can be isolated from natural materials, preferably plants. 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 from the Polygonum avicularis herba.

Specifically, the cell aging is characterized by aging of the fibroblasts or aging of the cells, wherein the aging of the fibroblasts is induced by adriamycin.

In addition, the inhibition of cell senescence is characterized in that inhibition of senescence-associated beta-galactosidase (SA-beta-gal) activity is measured.

In addition, the composition of the present invention may be provided in various forms selected from a pharmaceutical composition or a functional food composition.

When the composition of the present invention is a pharmaceutical composition, the pharmaceutical composition may include a pharmaceutically acceptable carrier other than the above-mentioned lauride [(-) - loliolide]. Such a pharmaceutically acceptable carrier may be a pharmaceutical preparation Starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like, which are conventionally used in the art. , Syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. 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.

The pharmaceutical composition may be administered according to the degree of symptoms of cell senescence. Usually, topical administration is preferred. 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 can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

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.

In the case of the food composition of the present invention, the kind of the food is not particularly limited. Examples of foods to which the above-mentioned lauride [(-) - loliolide] can be added include dairy products including meat, sausage, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, Various soups, drinks, tea, drinks, alcoholic beverages, and vitamin complexes.

The inventors of the present invention have confirmed that a compound of lauride [(-) - loliolide] isolated from an axon axis inhibits adiamycin-induced cell senescence and also inhibits cell senescence in cells induced by replication senescence. Thus, by inhibiting the cellular senescence process of human fibroblasts, 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 have.

Figure 1 shows the effect of loriolide [(-) - loliolide] on human fibroblasts on cell senescence by adriamycin. Cells were treated with adriamycin, treated with 10 ug / ml of lauride [(-) - loliolide], and stained with SA-β-gal activity 3 days later. A, SA-β-gal active staining in human fibroblasts; B, SA-β-gal active stain percentage in human fibroblasts. The results are typical photographs obtained after repeatedly performing each experiment three times or more independently. The results are expressed as mean and standard deviation. C, control group; D, dimethylsulfoxide; N, N-acetylcysteine; R, rapamycin. * p <0.05 or ** p <0.01 vs DMSO.
Figure 2 shows the effect of loriolide [(-) - loliolide] on the cellular senescence by adriamycin in umbilical vein endothelial cells. Cells were treated with adriamycin, treated with 10 ug / ml of lauride [(-) - loliolide], and stained with SA-β-gal activity 3 days later. A, SA-β-gal active staining in human umbilical vein endothelial cells; B, Percentage of SA-β-gal active staining in human umbilical vein endothelial cells. The results are typical photographs obtained after repeatedly performing each experiment three times or more independently. The results are expressed as mean and standard deviation. C, control group; D, dimethylsulfoxide; N, N-acetylcysteine; R, rapamycin. * p <0.05 or ** p <0.01 vs DMSO.
FIG. 3 shows the cell aging inhibitory effect of the lauride [(-) - loliolide] according to the concentration in human fibroblasts. After treatment with adriamycin, SA-β-gal activity staining was performed by treatment with loriolide [(-) - loliolide] in a concentration-dependent manner. A, SA-β-gal active stain pictures and percentages; B, SA-β-gal active staining percentage. The results are typical photographs obtained after repeatedly performing each experiment three times or more independently. The results are expressed as mean and standard deviation. C, control group; D, dimethylsulfoxide; N, N-acetylcysteine; R, rapamycin. * p <0.05 or ** p <0.01 vs DMSO.
Figure 4 shows the results of active oxygen control by the loriolide [(-) - loliolide] in human fibroblasts. After adriamycin treatment, the loriolide [(-) - loliolide] was treated in a concentration dependent manner and the amount of active oxygen was examined by flow cytometry using DCF fluorescence. A, flow cytometry results; B, the median fluorescence. Results were expressed as means and standard deviations obtained after repeatedly performing each experiment three or more times independently. NT, except for adriamycin; ADR, adriamycin; C, control group; D, dimethylsulfoxide; N, N-acetylcysteine; R, rapamycin. * p < 0.05 or ** p < 0.01 vs DMSO
Fig. 5 shows the results of regulating p53 protein expression by the loriolide [(-) - loliolide] in human fibroblasts. After treatment with adriamycin, the treatment with loriolide [(-) - loliolide] was performed in a concentration-dependent manner, and the degree of expression of p53 protein was examined by Western blotting. The results are typical photographs obtained after repeatedly performing each experiment three or more times independently. NT, except for adriamycin; ADR, adriamycin; C, control group; D, dimethylsulfoxide; N, N-acetylcysteine; R, rapamycin.
Fig. 6 shows the cell aging inhibitory effect of the lauride [(-) - loliolide] in human fibroblasts induced by replication senescence. We examined the changes of SA-β-gal activity in old cells induced by replication-induced senescence by concentration of lauride [(-) - loliolide]. A, SA-β-gal active staining; B, SA-β-gal active stain percentage. The results are typical photographs obtained after repeatedly performing each experiment three times or more independently. The results are expressed as mean and standard deviation. O, Old cells; D, dimethylsulfoxide; N, N-acetylcysteine; R, 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> Laurie Ride [(-) - loliolide ] Separation and structure determination

1. Isolioleide [(-) - loliolide] Isolation

9 kg of dried shrimp was extracted with 70%, 90%, 100% MeOH (13 L, once) at refluxing temperature of about 60 ° C for 24 hours and the total extract was concentrated under reduced pressure to obtain 1.4 kg of MeOH extract (PA1). The distilled water in MeOH extract (1.4 L) and n - hexane layer was distilled fraction funnel was added to (n -Hexane) (1.4 L) and n - hexane 3 times the operation of the fraction (n -Hexane) layer, and each fraction extracted with hexane (n -Hexane) was obtained the extract, the number of layers again in the same manner as in EtOAc, BuOH order n - - the extract was concentrated under reduced pressure to distilled water and n-hexane (n -Hexane) extract (PA2, 120 g ), EtOAc extract (PA3, 65 g), BuOH extract (PA4, 140 g) and H ₂ O extract (PA5, 800 g) were obtained.

From these fractions, 50 g of EtOAc extract (PA3) was subjected to normal phase column chromatography. Columns of silica gel (100 x 11 cm) (silica gel; No.9385, 230-400 mesh, Merck) to fill approximately 30 cm n - hexane (n -Hexane) eluted (elution) in 3 L fixed bed (stationary phase ) Was homogenized. Then, 50 g of the sample was adsorbed on 200 g of silica gel (No. 7734, 70-230 mesh, Merck) and loaded on the column. After n - a - (hexanes gradient from 100% to EtOAc 100% n), EtOAc- MeOH 29 fractions (PAE1 ~ 29) eluting with (gradient to 100% MeOH in EtOAc 100%) hexane (Hexane) -EtOAc . Elution with MeOH-H ₂ O (20: 80 gradient in 100% MeOH) on a reverse-phase column (4 x 50 cm, LiChroprep RP-18) in PAE12 of the fraction yielded the lauride [(-) - loliolide; PAC5, 20 mg] was obtained, which was dissolved in dimethyl sulfoxide and treated with cells.

2. Physico-chemical properties of lauride [(-) - loliolide]

The structure was identified by comparing the following spectroscopic analysis data with the literature data (Kor. J. Pharmacogn. (2007) 38: 291-295), which was isolated from the monocotyledonous extract [(-) - loliolide] ). Spectroscopic data are as follows.

_{A colorless gum C 11 H 16 O} 3 ¹ H-NMR (250 MHz, methanol-d ₄ ) 4.22 (1H, brq, J = 2.5 Hz, H-6), 2.45 (1H, dt, J = 12.5 and 2.5 Hz, H- , J = 12.5 and 2.5 Hz, H-7), 1.76 (3H, s, H-10), 1.73 (1H, dd, J = 13.8 and 3.9 Hz, H-5), 1.51 (1H, dd, J = 13.8 and 2.5 Hz, H-7 ), 1.47 (3H, s H-8), 1.29 (3H, s, H-9) 13 CNMR (62.9 MHz, Methanol-d 4) 185.9 (C-3α, 171.6 (C 2), 113.5 (C-3), 89.1 (C-7 ?, 67.4 (C-6), 48.1 (C-5), 46.6 ), 27.6 (C-9), 27.1 (C-10) Positive FABMS m / z 197 [M + H] ⁺ [? ²⁵ _D : -122.2 (c 0.09 MeOH).

&Lt; Formula 1 >

< Example  2> human fibroblasts In umbilical vein endothelial cells  Investigation of inhibition of cellular senescence of [(-) - loliolide]

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 p21 and p53 were obtained from Santa Cruz Biotech, Inc. (SantaCruz, CA, USA), and antibodies against pS6 were purchased from Cell Signalin Technology Inc. (Beverly, MA, USA). GAPDH antibody was distributed by Dr. Ki-sun Kwon, Korea Research Institute of Bioscience and Biotechnology. Adriamycin used products of Ildong Pharmaceutical Co., Ltd.

2. Cell culture

Human fibroblasts and umbilical vein endothelial cells were used as the cells. Human fibroblasts were cultured in a DMEM culture medium containing 10% fetal bovine serum and 1% antibiotic (penicillin 10,000 unit / ml, streptomycin 10,000 mg / ml) in a 100 mm diameter culture dish at 1 × 10 ⁵ cells And then cultured in a 5% carbon dioxide incubator at 37 ° C. When 80-90% of the cells were grown on the bottom of the culture dish, the cells were treated with trypsin-EDTA solution (2.5%), and subcultured. The umbilical vein endothelial cells were cultured in the same manner using EGM-2 as the culture medium. The number of cells was measured every time the cells were transplanted to examine how many times the cells were disrupted. The population doubling (PD) was examined by the following equation. PD = log ₂ F / log ₂ I (F = last cell number, I = first 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 umbilical vein endothelial cells.

3. Induction of Cell Aging by Adriamycin Treatment

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

4. Investigation of the effects of single compounds on cell senescence by adriamycin

We examined the effect of single compounds on the cell aging induced by adriamycin. Cells treated with adriamycin for 4 hours were separated from the culture dish with trypsin-EDTA. Cells were cultured in 96-well cell culture dishes in a DMEM culture medium containing 10% fetal bovine serum and 1% antibiotic, and 5,000 cells / ml, and EGM-2 culture medium, the vascular endothelial cells of the umbilical vein were 10,000 cells / ml, and 100 ml of the cells were dispensed into each well. Finally, 500 fibroblasts and 1,000 umbilical vein endothelial cells were dispensed per well. Incubated at 37 ° C., 5% carbon dioxide incubator for one day. After adding 100 ml of DMEM medium containing 10% fetal bovine serum and 1% antibiotic and EGM-2 culture medium to each well, 10 mg / ml of the single compound was treated. Dimethyl sulfoxide was added as a negative control and 5 mM of N-acetylcysteine and 500 nM of 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.

5. MTT measurement method

The degree of cell growth was evaluated by measuring the concentration of 3- (4,5-dimethylthiazol-2-yl) -2,5-dimethylthiazol-2yl-2,5-diphenyltetrazolium bromide (MTT ) Method. 50 μl of 0.1% MTT solution was added to each well of a 96-well culture container and reacted in a 5% carbon dioxide incubator at 37 ° C for 3 hours. After removing the culture solution and the MTT solution, 100 ul of dimethyl sulfoxide was added to dissolve the crystals formed. Absorbance was measured at 550 nm using a microplate reader.

6. Senescence-associated β-galactosidase (SA-β-gal) activity staining

The effect of single components on cell senescence was investigated with SA-β-gal active staining. After treating each single component in a 24 well culture vessel or a 12 well culture vessel for 3 days, the cells were washed with phosphate buffer. Cells were fixed with 3.7% paraformaldehyde and stained with 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 MgCl2, X-gal 1 mg / ml] , And 500 ul were added to 12 wells. Wrapped in silver foil, and reacted at 37 ° C for 16 hours to 18 hours. After washing twice with phosphate buffer (PBS), it was stained for 1 minute with 1% Eogene solution. After washing twice with phosphate buffer solution, the cells stained blue with an optical microscope were observed. SA-β-gal activity level was expressed as a percentage (%) by measuring the number of cells stained blue in the cytoplasm out of a total of 50-100 cells.

7. Cell Protein Extraction

Each cell was dispensed into 1 × 10 ⁵ in a 60 mm culture dish and then cultured in a 37 ° C., 5% carbon dioxide incubator. The cells were washed twice with DMEM medium containing antibiotics, and the fraction of the monocotyledonous extract and the compound was treated for 1 hour before the concentration and the adriamycin 500 nM for 4 hours. After the culture solution was removed, it was washed once with phosphate buffer solution. Cell lysis 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, 5 mM NaF, protease inhibitor or 1 mM PMSF]. The solution and cells were collected using a cell scrape and then transferred to a micro-incision tube. The solution was shaken every 10 minutes while reacting for 30 minutes on ice. The supernatant was transferred to a new tube by spinning at 12,000 xg for 10 min. 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 isolated by electrophoresis on 10% SDS-polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and allowed to react for 1 hour in Tween-20-Tris buffered saline (TTBS) containing 5% whole milk powder. The nitrocellulose membrane was reacted overnight in a 5% whole milk powder-TTBS solution containing primary antibodies against p53 or p21. After washing three times with TTBS solution, they were reacted with a secondary antibody conjugated with horseradish peroxidase for 3 hours. The membranes were washed five times for 5 min with TTBS, and then the amount of p53, p21 and pS6 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 confirmed using glyceraldehyde-3-phosphate dehydrogease (GAPDH) antibody.

9. Determination of free radicals in cells

Each cell was divided into 1.5x10 ⁵ cells in a 100 mm culture dish and cultured in a 5% CO 2 incubator at 37 ° C for 3 days. After washed twice with a DMEM cell culture medium containing the antibiotics, adriamycin handle 500 nM for 4 hours and separating the cells by treatment with trypsin -EDTA solution (2.5%), 1 X 10 5 to 60mm petri dishes The dog was again busted. Incubated at 37 ° C., 5% carbon dioxide incubator for one day. The culture was changed and treated with 10 ug / ml of laurylide [(-) - loliolide]. Dimethyl sulfoxide was added as a negative control and 5 mM of N-acetylcysteine and 500 nM of rapamycin were added as a positive control. After incubation for 3 days at 37 ℃, 5% carbon dioxide incubator, washed twice with DMEM culture medium containing antibiotics and was treated with H ₂ DCFDA 250uM for 20 minutes. The cells were washed twice with phosphate buffered saline and treated with trypsin-EDTA solution (2.5%) to transfer the cells to the microcirculation tube. The supernatant was discarded at 12,000 xg for 10 minutes, and the cells were washed with 1 ml of phosphate buffer solution containing 2% fetal bovine serum, and further centrifuged at 12,000 xg for 10 minutes. After repeated washing twice, 1 ml of 1% paraformaldehyde was added. ROS was measured using a BD FACS Canto II flow cytometer (BD Biosciences, San Jose, Calif.).

10. Statistical processing

Each experiment was repeated three times at three times at a time to obtain mean and standard deviation. Statistical significance was analyzed by Student's t-test, and p value <0.05 (p <0.05) was considered significant.

11. Results

At first, cytotoxicity was not observed at the concentration of 10 ug / ml when the loriolide [(-) - loliolide] was cytotoxic in human fibroblasts and umbilical vein endothelial cells. The degree of inhibition of cell senescence by the laurylide [(-) - loliolide] was investigated by SA-β-gal activity staining well known as a cell senescence marker. And compared with N-acetylcysteine (NAC) and rapamycin, which have been reported to be effective in inhibiting cell senescence. First, adriamycin-treated cells were treated with 10 ug / ml of lauride [(-) - loliolide], and the degree of senescence was compared with SA-β-gal staining after 3 days. As a result, in human fibroblasts, SA-β-gal active staining increased by adriamycin treatment was reduced by treatment with lorolides [(-) - loliolide], but not in umbilical vein endothelial cells 1 and Fig. 2). Therefore, only the human fibroblasts were included in the subsequent experiments. (-) - loliolide] was investigated for the concentration - dependent inhibition of cellular senescence. As a result, it was confirmed that the SA-β-gal activity increased by adriamycin was decreased as the concentration of the lauride [(-) - loliolide] was increased (FIG.

We investigated the effect of adriamycin on the amount of active oxygen increased by further investigating the inhibitory effect of loriolide [(-) - loliolide] on cell senescence. As a result, it was observed that the lauride [(-) - loliolide] also decreased the active oxygen that is increased by adriamycin (Fig. 4). It is well known that the expression of p53, one of the cancer suppressor genes, is increased during cell aging by adriamycin (Mol Biol Cell. 2007; 18: 4543-4552). Therefore, we investigated how the expression of p53, which is increased by adriamycin by the loriolide [(-) - loliolide], changes by the Western blot method. As a result, increasing the concentration of the loriolide [(-) - loliolide] did not decrease the expression of p53, which is increased by adriamycin (Fig. 5). From the above results, it was confirmed that the loriolide [(-) - loliolide] inhibited adriamycin - induced cell senescence in fibroblasts, unlike the umbilical vein endothelial cells of human umbilical vein.

On the other hand, it was investigated whether or not the loriolide [(-) - loliolide] can regulate not only the cell aging induced by adriamycin but also the cell senescence of the reproductive aging cells. Human fibroblasts induced transcriptional senescence through subculture, and SA-β-gal activity was examined in aged cells with increasing concentrations of lauride [(-) - loliolide]. As a result, it was observed that the loriolide [(-) - loliolide] decreased the SA-β-gal activity, which was increased in old cells in a concentration-dependent manner (FIG. From the above results, it was confirmed that the roriolide [(-) - loliolide] also suppressed the replication senescence. Thus, it has been observed that the loriolide [(-) - loliolide] has the effect of inhibiting aging not only in adipocyte-induced cell senescence in human fibroblasts but also in replicating senescent cells.

Claims (9)

A composition for inhibiting cell aging containing loriolide [(-)-loliolide] represented by the following Formula 1 as an active ingredient.

&Lt; Formula 1 >

The composition of claim 1, wherein the loriolide [(-)-loliolide] is isolated from an extract of Polygoni avicularis herba. The method of claim 2, wherein the extract is Single shaft Single shaft (Polygoni avicularis herba) A composition for inhibiting cell aging, characterized in that fractionation is performed by adding ethyl acetate (EtOAc) to the distilled water layer fractionated by adding distilled water and hexane (n-hexane) to the methanol extract. According to claim 1, wherein the cell aging is a composition for inhibiting cell aging, characterized in that aging or replication aging of fibroblasts. The composition of claim 4, wherein the aging of the fibroblasts is induced by adriamycin. The composition of claim 1, wherein the inhibition of cellular senescence measures inhibition of senescence-associated β-galactosidase (SA-β-gal) activity. The composition for inhibiting cell senescence according to claim 1, wherein the composition is a pharmaceutical composition. The method of claim 7, wherein the pharmaceutical composition is characterized in that any one selected from the group consisting of skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin damage tissue, arteriosclerosis, prostate hyperplasia and liver cancer Cell aging inhibitory composition. According to claim 1, wherein the composition is a composition for inhibiting cell aging, characterized in that the food composition.

Images