WO2014038872A1

WO2014038872A1 - Composition for inhibiting cellular senescence comprising loliolide

Info

Publication number: WO2014038872A1
Application number: PCT/KR2013/008030
Authority: WO
Inventors: Jae-Ryong Kim; Jong-Keun Son; Hyo-Hyun YANG; Kyoung HWANGBO
Original assignee: Industry Academic Cooperation Foundation of Yeungnam University
Current assignee: Industry Academic Cooperation Foundation of Yeungnam University
Priority date: 2012-09-06
Filing date: 2013-09-05
Publication date: 2014-03-13
Anticipated expiration: 2015-03-06
Also published as: KR101437729B1; KR20140032201A

Description

[DESCRIPTION]

[Invention Title]

COMPOSITION FOR INHIBITING CELLULAR SENESCENCE COMPRISING LOLIOLIDE

[Technical Field]

The present disclosure relates to a composition for inhibiting cellular senescence comprising loliolide [(-)-loliolide] as an active ingredient. [Background Art]

Cellular senescence is the phenomenon by which normal somatic cells cease to divide, after a certain number of cell divisions. This phenomenon is also called replicative senescence because it is induced by DNA damage resulting from gradually shortened telomere of the end of a chromosome during cell division (Collado et al., Cell. (2007) 130:223-233). The cellular senescence is also induced by not only telomere shortening, but also dysfunction of oncogenes and tumor suppressor genes, inflammatory reaction, oxidative stress, anti-cancer drugs, UV, radioactive rays and the like (Kuilman et al., Genes & Development. (2010) 24:2463- 2479). Senescent cells have characteristic such as enlarged and flattened morphology, growth arrest, DNA damages in nucleus and secreted various inflammatory proteins (Rodier and Campisi, J Cell Biol. (2011 ) 192:547-556). And, it has been known that the senescence-associated β-galactosidase (SA- -gal) activity is biochemically increased (Dimri et al., Proc Natl Acad Sci U S A. (1995) 92:9363- 9367). It has been known that the senescence is induced by various factors, but the senescence is controlled through p53 and Rb/p16 tumor suppressor gene signaling pathway (Campisi, Curr Opin Genet Dev. (2011 ) 21 :107-112).

The cellular senescence phenomenon inhibits or promotes cancer, and is suggested as an important mechanism of tissue regeneration and repair, aging of tissues/organisms and aging-related diseases. In addition, the cellular senescence causes various aging-related diseases such as cancer, arteriosclerosis, skin aging, degenerative nerve diseases, sarcopenia, osteoporosis and prostatic hyperplasia. Recent studies report that when the cellular senescence is selectively controlled, aging of tissues and organs, health-adjusted life expectancy and developing aging- related diseases can be controlled. It has been known that in telomerase-deficient mice, aging is accelerated, and it was confirmed that the increased telomerase expression in old telomere-deficient mice reverses aging-associated degenerative changes in tissues or organs (Jaskelioff et al., Nature. (2011 ) 469:102-106). It was confirmed that when cells expressing p16, whose expression is known to be increased in senescent cells of a lifespan shortened mouse model, is selectively removed, senescence-induced lesions on tissues are inhibited, and generation of the aging-related diseases is reduced (Baker et al., Nature. (2011 ) 479:232-236). In mice, hepatic stellate cells are aged during hepatic fibrosis process, and it has been known that the aging of hepatic stellate cells inhibits excessive hepatic fibrosis. It has been known that too high p53 activity, without being properly controlled, accelerates senescence, but on the contrary, proper p53 activity inhibits senescence.

And, some study results about materials having cellular senescence inhibitory effect also reported. Drugs or single component such as vitamin C, N- acetylcysteine, NS398 and epifriedelanol inhibit the cellular senescence (Won-Sang et al., Nutrition Research and Practice. (2007) 1 :105-112; Kim et al., Mech Ageing Dev. (2008) 129:706-713; Yang et al., Planta Med. (2011 ) 77:441 -449). And, it was reported that rapamycin in a mouse model and 4,4'-diaminodiphenylsulfone in Caenorhabditis elegans inhibit generation of aging-related diseases, and expand health-adjusted life expectancy (Harrison et al., Nature. (2009) 460:392-395; Cho et al., Proc Natl Acad Sci U S A. (2010) 107:19326-19331 ).

Polygoni avicularis herba is also called knotgrass, and has antioxidant effect. It also known to have various effects such as: improving sperm mobility, which is reduced by electromagnetic wave exposure, in mouse model; recovering gum inflammation in human; inhibiting bile duct ligation-induced hepatic fibrosis; recovering acetaminophen-induced nephrotoxicity; and releasing vascular smooth muscle cells and thereby expanding blood vessels (Milan et al., Pak J Biol Sci. (2011 ) 14:720-724; Sohn et al., Environ Toxicol Pharmacol. (2009) 27:225-230; Yin et al., J Ethnopharmacol. (2005) 99:113-117).

On the other hand, the present inventors disclosed a pharmaceutical composition for inhibiting aging comprising herb extract, which is at least one selected from the group consisting of Rhei rhizoma, Cirsii Radix, Plantaginis semen, Cinnamoni cortex, Cinnamoni cortex spissus, Euonimi lignum suberalatu, Salicis radicis cortex, Polygoni avicularis herba and Chaenomelis langenariae radix, as an active ingredient in Korean Patent Publication No. 10-2011-0041710, but there was no mention about loliolide [(-)-loliolide] compound of the present disclosure.

[Disclosure]

[Technical Problem] The present disclosure is directed to providing a composition for inhibiting cellular senescence comprising loliolide [(-)-loliolide] as an active ingredient.

The present disclosure is also directed to providing a pharmaceutical composition for inhibiting cellular senescence comprising loliolide [(-)-loliolide] as an active ingredient, which may exert therapeutic effect for skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronically damaged skin tissue, arteriosclerosis, prostatic hyperplasia, liver cancer and the like.

The present disclosure is also directed to providing a use of loliolide [(-)- loliolide] for preparing the composition for inhibiting cellular senescence, or a method for inhibiting cellular senescence, which comprises a step of administrating therapeutically effective amount of loliolide [(-)-loliolide] into a subject.

[Technical Solution]

As one general aspect, in the present disclosure, cellular senescence inhibitory effect of 12 kinds of single component, isolated and purified from Polygoni avicularis herba extract, in human fibroblasts and umbilical vein endothelial cells was examined. As a result, the present disclosure was completed by founding that, of them, loliolide [(-)-loliolide] inhibits adriamycin-induced cellular senescence in human fibroblasts, and also inhibits cellular senescence in replicative senescence-induced cells.

The present disclosure provides a composition for inhibiting cellular senescence comprising loliolide [(-)-loliolide], represented by the following Chemical Formula 1 , as an active ingredient. Specifically, the loliolide [(-)-loliolide] may be isolated from Polygoni avicularis herba extract, and more specifically, the Polygoni _,

5

avicularis herba extract may be prepared by adding ethyl acetate (EtOAc) to a distilled water layer, which is fractionated after adding distilled water and hexane (n- hexane) to Polygoni avicularis herba methanol extract, and then fractionating thereof.

The loliolide [(-)-loliolide] of Chemical Formula 1 may be isolated from natural materials, specifically, plants. It may be isolated from various organs, roots, stems, leaves, flowers and plant tissue culture extracts of natural, cross and variety plants. The most specifically, it may be isolated from Polygoni avicularis herba.

Specifically, the cellular senescence may be senescence or replicative senescence of fibroblasts, and the senescence of fibroblasts may be induced by adriamycin.

Further, the cellular senescence inhibitory effect may be determined by measuring inhibition of senescence-associated β-galactosidase (SA- -gal) activity.

Further, the composition of the present disclosure may be provided in various forms selected from a pharmaceutical composition or a functional food composition.

When the composition of the present disclosure is a pharmaceutical composition, the pharmaceutical composition may contains pharmaceutically acceptable carriers other than the loliolide [(-)-loliolide], and the pharmaceutically acceptable carriers may be carriers generally used for formulating drugs, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil and the like, but not limited thereto. Further, the pharmaceutical composition may further contain additives such as lubricants, humectants, sweetening agents, flavoring agents, emulsifiers, suspending agents and preservatives.

A method for administrating the pharmaceutical composition may be determined according to the degree of cellular senescence, and generally, it may be a local administration method. Further, therapeutically effective amount of the active ingredient in the pharmaceutical composition may differ from administration route, severity of disease, age, gender and body weight of a patient, and the like, and for example, daily dosage may be 0.01 to 1 ,000 mg/kg, specifically 0.1 to 1 ,000 mg/kg, more specifically 0.1 to 100 mg/kg. The administration may be made once a day or several times a day.

The pharmaceutical composition may be administered into mammals as a subject including rat, mouse, cattle and human through various routes. All administration routes may be employed, for example, oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine, epidural or intracerebroventricular routes.

The pharmaceutical composition may be manufactured in a single-dose formulation or enclosed in a multiple-dose vial by formulating using pharmaceutically acceptable carriers and/or excipients. At this time, the formulation may be in the form of solutions, suspensions or emulsions, or elixirs, extracts, powders, granules, tablets, plaster, lotions or ointments.

On the other hand, the pharmaceutical composition may treat any one disease selected from the group consisting of skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronically damaged skin tissue, arteriosclerosis, prostatic hyperplasia and liver cancer, but not limited thereto.

Further, when the present disclosure is a food composition, the kind of the food may not be particularly limited. Examples of foods, to which the loliolide [(-)- loliolide] can be added, may include meats, sausages, breads, chocolates, candies, snacks, confectioneries, pizza, instant noodles, other noodles, gums, dairy products including ice cream, various soups, beverages, teas, drinks, alcoholic beverages and multi-vitamin preparations. [Advantageous Effects]

The present inventors confirmed that the loliolide [(-)-loliolide] compound isolated from Polygoni avicularis herba inhibits adriamycin-induced cellular senescence, and also inhibits cellular senescence in replicative senescence-induced cells. It may be usefully used for treating aging-related diseases, for example, skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronically damaged skin tissue, arteriosclerosis, prostatic hyperplasia and liver cancer, by inhibiting the cellular senescence process of human fibroblasts.

[Description of Drawings]

Fig. 1 represents the effect of the loliolide [(-)-loliolide] on adriamycin-induced cellular senescence in human fibroblasts. The adriamycin was treated to the cells, the loliolide [(-)-loliolide] was treated in an amount of 10 pg/ml, and then 3 days later, βΑ-β-gal activity staining was conducted (A, images of SA- -gal activity staining in human fibroblasts; B, percentage of δΑ-β-gal activity staining in human fibroblasts). The results were traditional images obtained after repeatedly conducting each experiment 3 or more times independently, and indicated by mean and standard deviation (C, control group; D, dimethyl sulfoxide; N, N-acetylcysteine; R, rapamycin. ^*p<0.05 or ^**p<0.01 vs DMSO).

Fig. 2 represents the effect of the loliolide [(-)-loliolide] on adriamycin-induced cellular senescence in umbilical vein endothelial cells. The adriamycin was treated to the cells, the loliolide [(-)-loliolide] was treated in an amount of 0 pg/ml, and then 3 days later, SA- -gal activity staining was conducted (A, images of SA- -gal activity staining in umbilical vein endothelial cells; B, percentage of βΑ-β-gal activity staining in umbilical vein endothelial cells). The results were traditional images obtained after repeatedly conducting each experiment 3 or more times independently, and indicated by mean and standard deviation (C, control group; D, dimethyl sulfoxide; N, N-acetylcysteine; R, rapamycin. ^*p<0.05 or ^**p<0.01 vs DMSO).

Fig. 3 represents the effect of the loliolide [(-)-loliolide] on cellular senescence depending on concentration in human fibroblasts. The adriamycin was treated to the cells, the loliolide [(-)-loliolide] was treated in a concentration-dependent manner, and then SA- -gal activity staining was conducted (A, images of SA- -gal activity staining; B, percentage of SA-p-gal activity staining). The results were traditional images obtained after repeatedly conducting each experiment 3 or more times independently, and indicated by mean and standard deviation (C, control group; D, dimethyl sulfoxide; N, N-acetylcysteine; R, rapamycin. ^*p<0.05 or ^**p<0.01 vs DMSO).

Fig. 4 represents the results of controlling reactive oxygen species by the loliolide [(-)-loliolide] in human fibroblasts. The adriamycin was treated to the cells, the loliolide [(-)-loliolide] was treated in a concentration-dependent manner, and then the amount of reactive oxygen species was examined by flow cytometry using DCF fluorescence (A, the results of flow cytometry; B, median of fluorescence). - The results were traditional images obtained after repeatedly conducting each experiment 3 or more times independently, and indicated by mean and standard deviation (NT, excluding adriamycin; ADR, adriamycin; C, control group; D, dimethyl sulfoxide; N, N- acetylcysteine; R, rapamycin. ^*p<0.05 or ^**p<0.01 vs DMSO).

Fig. 5 represents the results of controlling p53 protein expression by the loliolide [(-)-loliolide] in human fibroblasts. The adriamycin was treated to the cells, the loliolide [(-)-loliolide] was treated in a concentration-dependent manner, and then the degree of p53 protein expression was examined by western blotting. The results were traditional images obtained after repeatedly conducting each experiment 3 or more times independently (NT, excluding adriamycin; ADR, adriamycin; C, control group; D, dimethyl sulfoxide; N, N-acetylcysteine; R, rapamycin).

Fig. 6 represents the cellular senescence inhibitory effect of the loliolide [(-)- loliolide] in replicative senescence-induced human fibroblasts. The change on the SA- -gal activity depending on the loliolide [(-)-loliolide] concentration was examined in old cells, in which replicative senescence was induced through subculture (A, images of ΘΑ-β-gal activity staining; B, percentage of SA- -gal activity staining). The results were traditional images obtained after repeatedly conducting each experiment 3 or more times independently, and indicated by mean and standard deviation (O, Old cells; D, dimethyl sulfoxide; N, N-acetylcysteine; R, rapamycin. ^*p<0.05 or ^**p<0.01 vs DMSO). [Best Mode]

The examples and experiments will now be described. The following examples and experiments are for illustrative purposes only and not intended to limit the scope of the present disclosure.

<Example 1 > Isolation and Structure Determination of Loliolide [(-)-loliolide] 1. Isolation of Loliolide [(-)-loliolide]

Dried Polygoni avicularis herba 9 kg was extracted with 70%, 90% and 100% MeOH (13 L, one time) while refluxed and cooled at about 60°C for 24 hours, and the obtained entire extract was concentrated under reduced pressure, and thereby MeOH extract (PA1 ) 1.4 kg was obtained. Distilled water (1.4 L) and n-hexane (1.4 L) were added to the MeOH extract, and fractionated three times into distilled water layer and n-hexane layer by using a fraction funnel. Each fraction was concentrated under reduced pressure, and thereby distilled water extract and n-hexane extract were obtained. Again, the distilled water layer was extracted with EtOAc and BuOH in order, according to the above method, and thereby n-hexane extract (PA2, 120 g), EtOAc extract (PA3, 65 g), BuOH extract (PA4, 140 g) and H₂O extract (PA5, 800 g) were obtained.

Of these fractions, the EtOAc extract (PA3) 50 g was subjected to normal phase column chromatography. A column (100 x 1 1 cm) was filled with Silica gel (No.9385, 230-400 mesh, Merck) up to about 30 cm, and eluted with n-hexane 3 L, so as to make stationary phase homogeneous. Sample 50 g was adsorbed to silica gel (No.7734, 70-230 mesh, Merck) 200 g, and loaded into the column. Then, the I I column was eluted with n-hexane-EtOAc (graded from n-hexane 100% to EtOAc 100 %) and EtOAc-MeOH (graded from EtOAc 100% to MeOH 100 %), thereby 29 fractions (PAE1 -29) were obtained. Of these fractions, PAE12 was eluted with MeOH-H₂O (graded with 100% MeOH to 20:80) in a reverse-phase column (4x50 cm, LiChroprep RP-18), and thereby loliolide [(-)-loliolide; PAC5, 20 mg] was obtained. The loliolide was dissolved in dimethyl sulfoxide, and then treated to cells.

2. Physicochemical Characteristics of Loliolide [(-)-loliolide]

Structure of the loliolide [(-)-loliolide] isolated from Polygoni avicularis herba extract was identified by comparing the following spectroscopic analysis data with reference (Arch Pharm Res (2004) 27, 1029-2033.) (Chemical Formula 1 ). Spectroscopic analysis data was as follows.

A colorless gum CnH₁₆O₃; [a] ² _D ^s -31.3 (c 0.1 methanol); ¹ H-NMR (250 MHz, Methanol-^) δ 5.74 (1 H, s, H-3), 4.22 (1 H, m, H-6), 2.43 (1 H, dt, J =13.6, 2.0 Hz H- 7a), 1.96 (1 H, dt, J - 14.3, 3.4 Hz, H-5a), 1.75 (3H, s, H-10); 1.72 (1 H, m, H-7b), 1.55 (1 H, dd, J = 14.3, 3.6 Hz, Η-5β), 1.45 (3H, s, H-8), 1.26 (3H, s, H-9); ¹³C-NMR (62.9 MHz, Methanol-d₄) δ 185.8 (C-2), 174.6 (C-7b), 113.5 (C-3), 89.1 (C-7a), 67.4 (C-6), 48.1 (C-5), 46.6 (C-7), 37.3 (C-4), 31.2 (C-9), 27.6 (C-10), 27.1 (C-8); Positive FABMS m/z 197.1 [M+H]⁺; <Chemical Formula 1 >

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<Example 2> Examination of Cellular Senescence Inhibitory Effect of Loliolide [(-)-loliolide] in Human Fibroblasts and Umbilical Vein Endothelial Cells

1. Test Materials

Human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells

(HUVECs) were purchased from Lonza (Walkersvill, MD, USA). Dulbeccos- Modified Eagle's medium (DMEM), fetal bovine serum and an antibiotic solution (penicillin-streptomycin) were purchased from WelGene (Daegu, Korea), and endothelial cell growth medium-2 (EGM-2) was purchased from Lonza (Walkersvill, MD, USA). Antibodies against p21 and p53 were purchased from SantaCruz Biotech, Inc. (SantaCruz, CA, USA), and an antibody against pS6 was purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). GAPDH antibody was divided from Dr. Ki-sun Kwon (KRIBB, Korea). Adriamycin was purchased from lldong Pharmaceutical Co., LTD (Korea).

2. Cell Culture

Human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) were used as cells. The human fibroblasts were divided into a culture dish (Diameter: 100 mm) to the cell number of 1 *10⁵ with DMEM medium containing 10% fetal bovine serum and 1 % antibiotics (penicillin 10,000 unit/ml, streptomycin 10,000 mg/ml), and cultured at 37°C in a 5% C0₂ incubator. When the cells were grown to about 80 to 90% confluency, the cells were separated by treating trypsin- EDTA solution (2.5%) thereto, and then the cells were subcultured. The umbilical vein endothelial cells were cultured with EGM-2 medium according to the above method. Every time the cells were subcultured, the cell number was measured, and how many times the cells were divided was examined. The number of population doubling (PD) was calculated by the following equation: PD= log₂F/log₂l (F=the final population number, l=the initial population number). For experiments, the human fibroblasts having PD<35 or PD>75, and the umbilical vein endothelial cells having PD<30 or PD>50 were used.

3. Cellular Senescence Induction by Adriamycin Treatment

The human fibroblasts and the umbilical vein endothelial cells were divided into a culture dish (Diameter: 100 mm) to the cell number of 1.5x10⁵, respectively. The cells were cultured at 37°C in a 5% CO2 incubator for 3 days, and then the cell medium was removed. The cells were washed two times with DMEM medium containing antibiotics, and then treated with 500 nM adriamycin for 4 hours. The cells were washed three times with DMEM medium containing antibiotics. And then the human fibroblasts were cultured with DMEM medium containing 10% fetal bovine serum and 1% antibiotics, and the umbilical vein endothelial cells were cultured with EGM-2 medium. After 4 days, whether cellular senescence was induced or not was confirmed by senescence-associated β-galactosidase (SA- -gal) activity staining. 4. Examination of Effect of Single Compound on Adriamycin-induced Cellular

Senescence

Whether single compound was effective on adriamycin-induced cellular senescence or not was examined. The cells treated with the adriamycin for 4 hours were separated from the culture dish with trypsin-EDTA. The fibroblasts were made to the cell concentration of 5,000 cells/ml in DMEM medium containing 10% fetal bovine serum and 1 % antibiotics, and the umbilical vein endothelial cells were made to the cell concentration of 10,000 cells/ml in EGM-2 medium, and then 100 ml of them were divided into each well of a 96-well cell culture plate. Finally, 500 cells of the fibroblasts and 1 ,000 cells of the umbilical vein endothelial cells were divided into each well, respectively, and then cultured at 37°C in a 5% C0₂ incubator for 1 day. 100 ml of DMEM medium containing 10% fetal bovine serum and 1 % antibiotics and EGM-2 medium were further added into each well, respectively, and the Polygon/ avicularis herba single compound was treated thereto to the concentration of 10 mg/ml. Dimethyl sulfoxide as a negative control group, and N-acetylcysteine 5mM and rapamycin 500nM as positive control groups were added to the cells, respectively. Then, the cells were cultured at 37°C in a 5% CO2 incubator for 3 days. The degree of cell growth was examined by MTT assay, and the degree of cellular senescence was examined by senescence-associated β-galactosidase (δΑ-β-gal) activity staining assay.

5. MTT Assay

The degree of cell growth was measured by 3-(4,5-dimethylthiazol-2yl)-2,5- diphenyltetrazolium bromide (MTT) assay. 0.1 % MTT solution 50 μΙ was added to each well of a 96-well culture plate, and reacted at 37°C in a 5% C0₂ incubator for 3 hours. The medium and the MTT solution were removed, and then dimethyl sulfoxide 100 μΙ was added thereto so as to dissolve formed crystals. Absorbance at 550 nm was measured by using a microplate reader. 6. Senescence-Associated β-Galactosidase (SA-P-gal) Activity Staining The effect of single component on cellular senescence was examined by SAP-gal activity staining. Each single component was treated in a 24-well culture plate or a 12-well culture plate for 3 days, and then the cells were washed with phosphate buffer. After fixing the cells with 3.7% paraformaldehyde, 250 μΙ of SA-p-gal staining solution [40 mM citric acid/phosphate, pH 5.8, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCI, 2 mM MgCI₂, X-gal 1 mg/ml] for the 24-well and 500 μΙ of the SA- -gal staining solution for the 12-well were added to each well, respectively. The plates were wrapped with aluminum foil, and then reacted at 37°C for 16 hours to 18 hours. The cells were washed two times with phosphate buffer (PBS), and then stained with 1 % eosin solution for 1 min. The cells were washed two times with phosphate buffer, and then the cells stained blue were observed with an optical microscope. The degree of SA- -gal activity was measured by counting the number of the cells, whose cytosols were stained blue, out of the total of about 50 to 100 cells and displayed as percentage (%).

7. Cell Protein Extraction

Each cells were divided into a 60 mm culture dish to the cell number of 1x10⁵, and then cultured at 37°C in a 5% C0₂ incubator. The cells were washed two times with DMEM medium containing antibiotics, and the fractions of Polygoni avicularis herba extract and the compounds were pretreated thereto for 1 hour by concentration, followed by treating adriamycin 500 nM for 4 hours. The medium was removed, and then the cells were washed one time with phosphate buffer. Cell lysis solution [25mM Tris-HCI (pH 7.6), 150mM NaCI, 1 % Tryton X-100, 0.5% sodium deoxycholate, 0.1 % SDS, 1 mM Sodium vanadate, 5mM NaF, protease inhibitor or 1 mM PMSF] 50 μΙ was added thereto. Entire solution and cells were collected by using a cell lifter, and then transferred to a microcentrifuge tube. The tube was reacted on ice for 30 min while vortexing the solution every 10 min. The tube was centrifuged at 12,000 xg for 10 min, and then supernatant was transferred to a new tube. The amount of the protein in the solution was quantified by bicinchoninic acid (BCA) method (Pierce Biotechnology Inc., Rockford IL, USA) by using bovine serum albumin as a standard protein. 8. Western Blot Analysis

Protein (30 pg) was separated by being electrophoresed through a 10% SDS- polyacrylamide gel. The protein was transferred to a nitrocellulose membrane, and then reacted in Tween-20-Tris buffered saline (TTBS) containing 5% dry whole milk for 1 hour. The nitrocellulose membrane was reacted with 5% dry whole milk-TTBS solution containing primary antibody against p53 or p21 overnight. The membrane was washed three times with TTBS solution, and then reacted with a horseradish peroxidase-conjugated secondary antibody for 3 hours. The membrane was washed five times with TTBS for every 5 min, and then the amounts of p53, p21 and pS6 were measured by using an enhanced chemiluminescence solution. The amount of the specific protein reacted with each antibody was measured by using a LAS-3000 imaging system (Fujifilm Corp., Stanford, CT, USA). Whether the same amount of the protein was used in each experiment was confirmed by using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody. 9. Measurement of Intracellular Reactive Oxygen Species (ROS) Concentration

Each cells were divided into a 100 mm culture dish to the cell number of 1.5x10⁵, and then cultured at 37°C in a 5% CO₂ incubator for 3 days. The cells were washed two times with DMEM medium containing antibiotics, and then adriamycin 500 nM was treated thereto for 4 hours. The cells were separated by treating trypsin-EDTA solution (2.5%), and then divided again into a 60 mm culture dish to the cell number of 1 X 10⁵ followed by culturing at 37°C in a 5% CO₂ incubator. The medium was replaced, and then loliolide [(-)-loliolide] 10 μg/ml was treated to the cells. Dimethyl sulfoxide as a negative control group and N- acetylcysteine 5 mM and rapamycin 500 nM as positive control groups were added to the cells. The cells were cultured at 37°C in a 5% CO₂ incubator for 3 days, washed two times with DMEM medium containing antibiotics, and then treated with H₂DCFDA 250 μΜ for 20 min. The cells were washed two times with phosphate buffer, and separated by treating trypsin-EDTA solution (2.5%) followed by transferring to a microcentrifuge tube. The tube was centrifuged at 12,000 xg for 10 min, and then supernatant was discarded. The cells were washed with 2% fetal bovine serum- containing phosphate buffer 1 ml, and then centrifuged again at 12,000 xg for 10 min. The cells were washed two times as described above, and then 1 % paraformaldehyde 1 ml was added thereto. ROS was measured by using BD FACS Canto II flow cytometry (BD Biosciences, San Jose, CA). 0. Statistical analysis

3 sets of each experiment were repeated three times or more, and then mean and standard deviation were calculated. Statistical significance was analyzed using Student t-test, and p value less than 0.05 (p<0.05) was considered as significant.

11. Result

First of all, whether the loliolide [(-)-loliolide] has cytotoxicity on the human fibroblasts and the umbilical vein endothelial cells or not was examined by MTT assay, and as a result, cytotoxicity was not observed at the concentration of 10 pg/ml. The cellular senescence inhibitory effect of the loliolide [(-)-loliolide] was examined by activity staining of SA-P-gal, well known as a cellular senescence marker. And, it was compared with N-acetylcysteine (NAC) and rapamycin, reported to have the cellular senescence inhibitory effect. First of all, the loliolide [(-)-loliolide] 10 pg/ml was treated to the adriamycin-treated cells, and 3 day later, the degree of senescence was compared by SA-3-gal activity staining. As a result, in the human fibroblasts, the increased SA- -gal activity staining induced by the adriamycin treatment was reduced by the loliolide [(-)-loliolide] treatment, but in the umbilical vein endothelial cells, there was no change (Fig. 1 and Fig. 2). Thus, in the later experiments, only human fibroblasts were used as a subject. Whether the cellular senescence inhibitory effect of the loliolide [(-)-loliolide] is concentration-dependent or not was examined. As a result, it was confirmed that the increased SA^-gal activity induced by adriamycin was reduced as the loliolide [(-)-loliolide] concentration was increased (Fig. 3).

In order to further examine the cellular senescence inhibitory effect of the loliolide [(-)-loliolide], how it affects to the amount of reactive oxygen species increased by adriamycin was examined. As a result, it was observed that the loliolide [(-)-loliolide] reduced the amount of the reactive oxygen species increased by adriamycin (Fig. 4). It has been reported that the expression of p53, one of tumor suppressor genes, is increased in the cellular senescence process induced by adriamycin (Mol Biol Cell. 2007;18:4543-4552). Thus, how the expression of p53 increased by adriamycin is changed by the loliolide [(-)-loliolide] was examined by western blot analysis. As a result, the expression of p53 increased by adriamycin was not reduced as the loliolide [(-)-loliolide] concentration was increased (Fig. 5). From the above results, it was confirmed that the loliolide [(-)-loliolide] inhibits the adriamycin-induced cellular senescence in the human fibroblast, unlike the human umbilical vein endothelial cells.

On the other hand, whether the loliolide [(-)-loliolide] controls the cellular senescence of replicative senescent cells as well as the adriamycin-induced cellular senescence or not was examined. In the human fibroblasts, the replicative senescence was induced through subculture, and then the SA-P-gal activity was examined while increasing the loliolide [(-)-loliolide] concentration in old cells. As a result, it was observed that the loliolide [(-)-loliolide] reduced the increased δΑ-β-gal activity in the old cells in a concentration dependent manner (Fig. 6). From the above results, it was confirmed that the loliolide [(-)-loliolide] also inhibits the replicative senescence. Thus, it was observed that the loliolide [(-)-loliolide] has the effect on inhibiting the senescence in the replicative senescent cells, as well as the adriamycin-induced cellular senescence in the human fibroblasts.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present disclosure. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the disclosure as set forth in the appended claims.

Claims

[CLAIMS]

[Claim 1 ]

A composition for inhibiting cellular senescence comprising loliolide [(-)- loliolide] represented by the following Chemical Formula 1 as an active ingredient.

[Claim 2]

The composition for inhibiting cellular senescence according to claim 1 , wherein the loliolide [(-)-loliolide] is isolated from a Polygoni avicularis herba extract.

[Claim 3]

The composition for inhibiting cellular senescence according to claim 2, wherein the Polygoni avicularis herba extract is prepared by adding ethyl acetate (EtOAc) to a distilled water layer, which is fractionated after adding distilled water and hexane (n-hexane) to Polygoni avicularis herba methanol extract, and then fractionating thereof.

[Claim 4]

The composition for inhibiting cellular senescence according to claim 1 , wherein the cellular senescence is senescence or replicative senescence of fibroblasts.

[Claim 5]

The composition for inhibiting cellular senescence according to claim 4, wherein the senescence of fibroblasts is induced by adriamycin.

[Claim 6]

The composition for inhibiting cellular senescence according to claim 1 , wherein the effect of inhibiting cellular senescence is determined by measuring inhibition of senescence-associated β-galactosidase (SA- -gal) activity.

[Claim 7]

The composition for inhibiting cellular senescence according to claim 1 , which is a pharmaceutical composition.

[Claim 8]

The composition for inhibiting cellular senescence according to claim 7, wherein the pharmaceutical composition is for treating any one disease selected from the group consisting of skin aging, rheumatoid arthritis, osteoarthritis, hepatitis, chronically damaged skin tissue, arteriosclerosis, prostatic hyperplasia and liver cancer.

[Claim 9]

The composition for inhibiting cellular senescence according to claim 1 , which is a food composition.

[Claim 10]

A use of loliolide [(-)-loliolide] represented by the following Chemical Formula 1 for preparing a composition for inhibiting cellular senescence.

[Claim 11 ]

A method for inhibiting cellular senescence comprising a step of administrating therapeutically effective amount of loliolide [(-)-loliolide] represented by the following Chemical Formula 1 into a subject in need thereof.