Arch. Pharm. Res. DOI 10.1007/s12272-015-0577-8

RESEARCH ARTICLE

Inhibitory effects of quercetagetin 3,40 -dimethyl ether purified from Inula japonica on cellular senescence in human umbilical vein endothelial cells Hyo Hyun Yang • Haiyan Zhang • Jong-Keun Son Jae-Ryong Kim



Received: 30 July 2014 / Accepted: 17 February 2015 Ó The Pharmaceutical Society of Korea 2015

Abstract Cellular senescence contributes to tissue and organismal aging, tumor suppression and progress, tissue repair and regeneration, and age-related diseases. Thus, aging intervention might be beneficial for treatment and prevention of diverse age-related diseases. In the present study, we investigated whether four compounds purified from Inula japonica exert inhibitory activity against cellular senescence induced by adriamycin in human umbilical vein endothelial cells (HUVECs). Among them, compound 4 (quercetagetin 3,40 -dimethyl ether) showed inhibitory activity against cellular senescence, which was confirmed by senescence-associated b-galactosidase (SAb-gal) activity, p53 and p21 protein levels, and intracellular ROS levels. Compound 4 also reduced SA-b-gal activity in HUVECs under replicative senescence. These results suggest that compound 4 represses cellular senescence in HUVECs and might be useful for the development of dietary supplements or cosmetics that alleviate tissue aging or age-related diseases.

Hyo Hyun Yang and Haiyan Zhang have contributed equally to this work. H. H. Yang  J.-R. Kim (&) Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu 705-717, Republic of Korea e-mail: [email protected] H. H. Yang  J.-R. Kim Aging-Associated Vascular Disease Research Center, College of Medicine, Yeungnam University, Daegu 705-717, Republic of Korea H. Zhang  J.-K. Son College of Pharmacy, Yeungnam University, Gyongsan 712-749, Republic of Korea

Keywords Cellular senescence  Inula japonica  Quercetagetin 3,4’-dimethyl ether  Human umbilical vein endothelial cells  Aging intervention

Introduction Normal somatic cells show a finite amount of cell proliferation, then undergo cellular senescence (Hayflick and Moorhead 1961). A variety of factors, including telomere attrition and dysfunction (Collado et al. 2007), activation of oncogenes and tumor suppressor genes, oxidative stress, inflammation, chemotherapeutic agents, and exposure to UV irradiation and ionizing radiation (Kuilman et al. 2010) have been reported to cause cellular senescence. Senescent cells have diverse characteristics, including flattened and enlarged cell morphology, senescence-associated b-galactosidase (SA-b-gal) activity, DNA damage foci in the nucleus, and senescence-associated secretory phenotypes (Rodier and Campisi 2011; Dimri et al. 1995). Accumulating evidence implies that cellular senescence contributes to tissue and organismal aging, tissue repair and regeneration, and cancer progression and protection. Cellular senescence is also involved in the pathogenesis of diverse age-related diseases, including cancer, skin aging, atherosclerosis, neurodegeneration, sarcopenia, benign prostate hyperplasia, and osteoporosis (Rodier and Campisi 2011). Recent evidence suggests that elimination of senescent cells or suppression of cellular senescence in vivo can prevent or reduce age-related tissue failure and increase health span. Removal of p16INK4a-positive senescence cells from progeroid mice delayed onset of age-related tissue phenotypes and retarded the progression of already established age-associated disorders (Baker et al. 2011). Additionally, restoration of telomerase in telomerase null mice to reveal premature aging

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phenotypes rescued age-related degenerative phenotypes (Jaskelioff et al. 2011). Several plant extracts and chemicals have been reported to exert inhibitory effects against cellular senescence. Some extracts prepared from medicinal plants used in East Asia, such as Rhei Rhizoma, Cirsii Radix, Plantagnis Semen, Cinnamomi Cortex, Euonymi Lignum Suberalatum, Salicis Radicis Cortex, Polygoni aviculari Herba, Chaenomelis langenariae Radix (Yang et al. 2010), Panax ginseng, Panax notoginsen, and Ligusticum chuanxiong (Yang et al. 2009; Im et al. 2012), are known to inhibit cellular senescence in human primary cells. Ascorbic acid (Hwang et al. 2007), N-acetylcysteine and NS398 (Kim et al. 2008), epifriedelanol (Yang et al. 2011), quercetin-3O-b-D-glucuronide (Yang et al. 2014b), juglanin (Yang et al. 2014c), and (-)-loliolide (Yang et al. 2014a) have also been reported to repress cellular senescence in human primary cells. The extracts of I. japonica flowers have long been used for treatment of inflammatory diseases in traditional medicine in East Asia (Park et al. 2011). These extracts have been found to contain sesquiterpenoids (Gong et al. 2011), diterpenoids (Qin et al. 2009), monoterpenes (Zhu et al. 2011), and flavonoids (Qin et al. 2010) and reported to have anti-diabetic (Shan et al. 2006), anti-asthmatic (Park et al. 2011), and anti-allergic (Lu et al. 2012) activities in animal models. However, the effects of individual compounds purified from I. japonica on cellular senescence have not been reported to date. In this study, we determined the chemical structure of four single compounds purified from I. japonica and investigated their inhibitory effects on cellular senescence in human umbilical vein endothelial cells (HUVECs). We found that compound 4 (quercetagetin 3,40 -dimethyl ether) exhibited inhibitory activity against cellular senescence in HUVECs.

Sun Kwon (KRIBB, Daejeon, Republic of Korea). Adriamycin was obtained from Ildong Pharmaceutical Co., Ltd. (Seoul, Republic of Korea). N-acetylcysteine (purity, [99 %) and rapamycin were purchased from SigmaAldrich Chemical Co. (St. Louis, MO, USA) for use as positive controls. Melting points were determined using a Fisher–Johns melting point apparatus and reported uncorrected. Optical rotations were measured using a JASCO DIP-1000 automatic digital polarimeter (JASCO, Tokyo, Japan). The NMR spectra were recorded on a Bruker 250 MHz spectrometer (Bruker Corp., Rheinstetten, Germany) using Bruker’s standard pulse program. Samples were dissolved in Methanol-d4, with chemical shifts reported in ppm downfield from TMS. FABMS was obtained using a JEOL JMS700 spectrometer (JEOL, Tokyo, Japan). The stationary phases used for column chromatography (Silica-gel 60, 70–230 and 230–400 mesh, Lichroprep Rp18 gel) and TLC plates (Silica-gel 60 F254 and Rp-18 F254, 0.25 mm) were purchased from Merck KGaA (Darmstadt, Germany). An LC-10AD pump, SPD-10A detector, and Shim-Pack prep-ODS (20 mm9 250 mm) column (Shimadzu Corp., Kyoto, Japan) were used for preparative HPLC. All other chemicals and solvents were of analytical grade and used without further purification. Plant material Inula japonica flowers were purchased in February 2009 from folk medicine market, ‘‘Yak-ryong-si’’ in Daegu, Republic of Korea. The materials were confirmed taxonomically by Professor Ki-Hwan Bae, Chungnam National University, Daejeon, Republic of Korea, and a voucher specimen (YNPA-2009) was deposited in the College of Pharmacy, Yeungnam University. Isolation and chemical structure identification of compounds 1–4

Materials and methods Materials Human umbilical vein endothelial cells (HUVECs) and endothelial cell growth medium-2 (EGM-2) were purchased from Lonza (Walkersvill, MD, USA). Fetal bovine serum (FBS) and penicillin–streptomycin solution were obtained from WelGene (Daegu, Republic of Korea). Antibody against p53 (SC-126) was acquired from Santa Cruz Biotech. Inc. (Santa Cruz, CA, USA) and antibodies against p21 (2947S) and phosphorylated S6 kinase (pS6K) at serine 240 and 244 (5364S) were acquired from Cell Signaling Technology Inc. (Beverly, MA, USA). Rabbit polyclonal antibody against glyceraldehydes 3-phosphate dehydrogenase (GAPDH) was kindly provided by Dr. Ki-

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The flowers of Inula japonica Thunb. (10 kg) were extracted three times with 70 % ethanol by reflux for 24 h, after which the ethanol solution was evaporated to dryness (1.5 kg). The ethanol extract was then suspended in H2O and the resulting H2O layer was partitioned with n-hexane, ethyl acetate and n-butanol three times, successively. Next, the ethyl acetate extract (130 g) was loaded onto a silica gel column (100 cm 9 11 cm, Silica-gel 70–230 mesh, lot no. 9385), and the column was eluted with n-hexane–ethyl acetate (gradient from n-hexane to ethyl acetate) and then ethyl acetate–methanol (gradient from ethyl acetate to methanol). The eluent was combined based on TLC, giving 27 fractions (IJ 1–27). Fraction IJ-13 (10 g) was recrystallized with chloroform and methanol for 2 h to give compound 2 (5 g). Fraction IJ-10

Inhibitory effects of quercetagetin 3,40 -dimethyl ether purified from Inula japonica

(3.5 g) was chromatographed on a silica-gel column (100 cm 9 5 cm, silica-gel 70–230 mesh, lot no. 9385) with ethyl acetate–methanol (gradient from 60:40–0:100), affording compound 1 (2 g). Fractions IJ-24 (1 g) were chromatographed on a reverse-phase column (30 cm 9 2 cm, Lichroprep Rp-18 gel) with methanol–H2O (1:9–10:0), and the main fraction from the column was subjected to preparative HPLC to give compounds 3 (20 mg) and 4 (38 mg). The chemical structures of compounds purified from I. japonica were determined to be: 1, tomentosin (Bohlmann et al. 1978; Spring et al. 1991), 2, britanin (Ito and Iida 1981; Silva et al. 1992), 3, 1, 5-di-O-caffeoylquinic acid (Merfort 1992; Tolonen et al. 2002), and 4, quercetagetin 3,40 -dimethyl ether (Shilin et al. 1989) by comparison of melting points, optical rotation values, 1H- and 13CNMR, and mass spectral data (Fig. 1). Cell culture HUVECs in EGM-2 media were cultured under the same conditions. The number of population doublings (PDs) was monitored for further experiments, after which PD was calculated using the geometric equation: PD = log2F/log2I (F, final cell number; I, initial cell number). HUVECs in PD \ 30 were used for adriamycin-induced cellular senescence. HUVECs in PD [ 50 were used as old cells under replicative senescence (Yoon et al. 2004; Kim et al. 2007). Induction of cellular senescence by adriamycin treatment HUVECs in EGM-2 media were plated at 1.5 9 105 cells per 100 mm culture plate. After incubation at 37 °C in a

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Treatment with isolated compounds To determine if the four individual compounds from I. japonica would repress adriamycin-induced cellular senescence, adriamycin-treated cells were dissociated by trypsinization. HUVECs were then plated at 1000 cells/ well in 96 well plates, after which they were incubated at 37 °C under 5 % CO2 humidified air for 24 h and then further treated with 1 or 10 lg/ml of one of the isolated compounds (1–4), 0.5 % dimethyl sulfoxide, 5 mM N-acetylcysteine, or 500 nM rapamycin for 3 days. Cell toxicity and cellular senescence were then measured by an MTT assay and SA-b-gal activity staining, respectively. MTT [3-(4,5-dimethylthiazol-2yl)-2,5diphenyltetrazolium bromide] assay Cells were treated with 0.1 % MTT solution for 3 h, after which the media was discarded and the resulting formazan crystals were solubilized with 100 ll dimethyl sulfoxide. Viability was then assessed by measuring the absorbance at 550 nm using a microplate reader. Senescence-associated b-galactosidase (SA-b-gal) activity assay SA-b-gal activity was determined as previously described (Dimri et al. 1995). Cells were counterstained with eosin, a total of 100 cells were counted in five randomized fields, and the percentage of blue cells was calculated. Protein extraction

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5 % CO2 incubator for 3 days, cells were washed twice with DMEM containing 1 % antibiotics and then treated with 500 nM adriamycin for 4 h. Following three rinses with DMEM containing 1 % antibiotics, HUVECs in DMEM containing 10 % FBS and 1 % antibiotics were incubated in a 5 % CO2 incubator for 4 days. Adriamycininduced cellular senescence was confirmed by senescenceassociated b-galactosidase (SA-b-gal) activity staining.

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Fig. 1 Chemical structures of compounds (1–4) isolated from I. japonica

Cells were plated at 1 9 105 in 60 mm culture dishes and then incubated for 24 h, after which they were treated with 2.9 lM of compound 4 for 1 h prior to adriamycin treatment. After incubation for 4 h, the cells were lysed in 50 ll of ice-cold RIPA buffer (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 particulate debris was removed by centrifugation at 12,000 9g for 10 min at 4 °C, after which the protein concentration in the supernatant was quantified by the bicinchoninic acid method

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cpd 4 (μM) ADR Fig. 2 Effects of compound 4 from I. japonica on adriamycininduced cellular senescence in HUVECs. Cells treated with 500 nM adriamycin for 4 h were seeded at 1000 cells/well in 96 well plates. After treatment with increasing concentrations of compound 4, cells were incubated for 3 days and cellular senescence assessed by SA-bgal activity staining. a SA-b-gal activity staining (9100).

b Percentages of SA-b-gal positive cells. Pictures of representative SA-b-gal stains from three independent experiments are shown. Values are the means ± SDs from three independent experiments measured in triplicate. N-acetylcysteine was used as a positive control. ADR adriamycin; C control; D dimethyl sulfoxide; N 5 mM N-acetylcysteine. *p \ 0.05 or **p \ 0.01 versus D

(Pierce Biotechnology Inc., Rockford, IL) using bovine serum albumin as a standard.

29 lM of compound 4, 0.5 % dimethyl sulfoxide, 5 mM N-acetylcysteine, or 500 nM rapamycin for 3 days. Next, cells were washed twice with DMEM containing 1 % antibiotics, then treated with 250 lM 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCFDA) for 20 min at 37 °C in a 5 % CO2 incubator. The cells were then harvested by trypsinization, washed twice with PBS containing 2 % FBS, and suspended in 1 % paraformaldehyde (pH 7.4). Finally, the DCF fluorescence intensity of each population of 10,000 cells was measured using a BD FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA).

Western blot analysis Proteins (20 lg) were separated on 10 % SDS–polyacrylamide gels and then transferred to nitrocellulose membranes. Following blocking of membranes with Tween-20 Tris-buffered saline (TTBS) containing 5 % skim milk for 1 h, the membranes were incubated overnight with antibodies against p53, p21, pS6K, and GAPDH. The membranes were then washed three times in TTBS, after which horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies were applied for 1.5 h. Antigen–antibody complexes were detected using Western Blotting Luminol Reagent (Santa Cruz Biotech Inc., Santa Cruz, CA, USA). Images of membranes were obtained using a LAS-3000 imaging system (Fujifilm Inc., Stamford, CT, USA). Equal loading of proteins was verified using GAPDH antibody.

Statistical analysis All data in the present study are presented as the mean ± SD from three independent experiments conducted in triplicate. Statistical significance was measured by a paired Student’s t test. A p value \ 0.05 was considered statistically significant.

Measurement of intracellular ROS level

Results

Adriamycin-treated cells were seeded at 1.5 9 105 in 60 mm culture dishes and then incubated at 37 °C in a 5 % CO2 incubator for 24 h. The cells were then treated with

We initially measured the cytotoxic effects of four compounds from I. japonica in HUVECs by an MTT assay. No cytotoxicity was observed in response to 10 lg/ml of 1, 3,

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DCF fluorescence intensity (Log) Fig. 3 Effects of compound 4 on the levels of p53, pS6K, and p21 proteins, and intracellular ROS in HUVECs treated with adriamycin. a The levels of p53, pS6K, and p21 proteins. Cells were treated with increasing concentrations of compound 4 for 1 h prior to adriamycin treatment, then incubated for 4 h. The proteins were then extracted from cells and separated, after which their expression levels were analyzed by Western blotting. b Intracellular ROS levels. Cells treated with or without adriamycin were seeded in 60 mm culture dishes and incubated for 24 h. Following treatment of cells with 29 lM of compound 4, 0.5 % dimethyl sulfoxide, 5 mM

N-acetylcysteine, or 500 nM rapamycin for 3 days, cells were loaded with 250 lM H2DCFDA for 20 min, after which the DCF fluorescence intensity of each population of 10,000 cells was measured by flow cytometry. N-acetylcysteine and rapamycin were used as positive controls. Representative data of three independent experiments are shown. Values are the means ± SDs from three independent experiments. NT not treated with adriamycin; ADR adriamycin; NT not treated; C control; D dimethyl sulfoxide; N 5 mM N-acetylcysteine; R 500 nM rapamycin; cpd 4 compound 4. *p \ 0.05 versus D

and 4, or 1 lg/ml of 2 (data not shown). We next investigated whether the four compounds exerted inhibitory effects against cellular senescence in HUVECs treated with adriamycin based on the SA-b-gal activity (Dimri et al. 1995). Compound 4 induced a dose-dependent reduction in the increase of SA-b-gal activity in HUVECs treated with adriamycin (Fig. 2), while compounds, 1, 2, and 3 exerted no inhibitory activity. We further tested effects of compound 4 on the expression levels of p53 and p21 proteins in adriamycin-treated cells, since these proteins are also known to increase during cellular senescence (Kim et al. 2007). Treatment with compound 4 suppressed the levels of p53 and p21 proteins (Fig. 3a). The level of pS6K was also measured to confirm the inhibitory effects of

rapamycin on cellular senescence, since rapamycin is a well known inhibitor of mammalian target of rapamycin (mTOR) that represses the phosphorylation of ribosomal S6 kinase (Demidenko et al. 2009). Since intracellular ROS levels reportedly increase in cells during adriamycin-induced cellular senescence (Yang et al. 2011), we evaluated whether compound 4 reduced the increase in ROS induced by adriamycin treatment in HUVECs. As expected, compound 4 decreased intracellular ROS levels observed in HUVECs treated with adriamycin (Fig. 3b). Since compound 4 inhibited adriamycin-induced cellular senescence in HUVECs, we investigated whether it also reversed replicative senescence. The results revealed that compound 4 decreased the SA-b-gal activity in HUVECs under

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Fig. 4 Effects of compound 4 on replicative senescence of HUVECs. Old cells (3 9 104/ well) were seeded in six well plates and incubated with increasing concentrations of four for 3 days. Cellular senescence was assessed by SAb-gal activity staining. a SA-bgal activity staining (9100). b Percentages of SA-b-gal positive cells. Pictures representative of three independent experiments are shown (9100). Values are the means ± SDs from three independent experiments. O old cells; D dimethyl sulfoxide; N 5 mM N-acetylcysteine; R 500 nM rapamycin; cpd 4 compound 4. **p \ 0.01 versus D

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replicative senescence (Fig. 4). Taken together, these findings imply that compound 4 from I. japonica might be able to rescue adriamycin-induced senescence as well as replicative senescence in HUVECs.

Discussion In the present study, four compounds from I. japonica were screened for inhibitory effects on cellular senescence in HUVECs. The results indicated that compound 4 repressed premature senescence induced by adriamycin treatment and replicative senescence, which was confirmed by SA-bgal activity. The flowers of I. japonica have long been utilized in traditional medicine of East Asia for treatment of inflammatory diseases and digestive disorders (Park et al. 2011). Aqueous-extract of I. japonica flowers has been reported to have anti-diabetic and hypolipidemic effects in alloxaninduced diabetic mice (Shan et al. 2006), and to effectively improve bowel movements and stool output in experimentally constipated mice (Shan et al. 2010). Additionally, the extract of I. japonica is known to have antiasthmatic effects in ovalbumin-induced asthmatic mice that occur via a reduction in eosinophil recruitment, airway hyper-responsiveness, and Th2 cytokine and IgE levels

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(Park et al. 2011). The ethanol extract of flowers of I. japonica revealed anti-allergic effects in response to mouse bone marrow-derived mast cells in vitro and a passive cutaneous anaphylaxis animal model in vivo that occurred by inhibiting mast cell mediated allergic reaction and mast cell activation (Lu et al. 2012). Although anti-inflammatory and anti-allergic activities of the extracts from flowers of I. japonica have been reported, to the best of our knowledge, this is the first report of anti-senescence activity of compound 4 in human primary cells based on SA-b-gal activity. Although diverse factors are involved in the induction of cellular senescence, two tumor suppressor pathways, p53/ p21 and Rb/p16INK4a, are critical to the regulation of cellular senescence (Campisi 2011). Our data revealed that the increased expression of p53 and p21 proteins in response to adriamycin treatment were reduced by treatment with compound 4 (Fig. 3a). These findings suggest that the p53/p21 pathway might be involved in the anti-senescence activity of compound 4 in HUVECs. Reactive oxygen species (ROS) have been reported to function in the regulation of cellular senescence (Yang et al. 2011; Kuilman et al. 2010). Intracellular ROS levels are upregulated during both premature and replicative senescence, and treatment with antioxidants such as N-acetylcysteine is known to repress cellular senescence

Inhibitory effects of quercetagetin 3,40 -dimethyl ether purified from Inula japonica

(Kim et al. 2009). ROS also activate the DNA damage signaling cascade, which subsequently induces cellular senescence (Kim et al. 2009). The present study showed that compound 4 decreased ROS levels in adriamycintreated cells (Fig. 3b), implying that the inhibitory effects of compound 4 on cellular senescence might be due to its antioxidant activity. The effects of compound 4 were determined using an in vitro cellular model. Therefore, further study is necessary to elucidate the mechanism by which compound 4 might prevent cellular senescence and demonstrate that its inhibitory effects occur in vivo. In conclusion, we found that compound 4 purified from I. japonica exerted an inhibitory effect on cellular senescence in HUVECs. This compound may be a promising candidate for development of dietary supplements or cosmetics to modulate tissue aging or aging-associated diseases. Acknowledgments This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (2005-0049417). Conflict of interest declare.

The authors have no conflicts of interest to

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Inhibitory effects of quercetagetin 3,4'-dimethyl ether purified from Inula japonica on cellular senescence in human umbilical vein endothelial cells.

Cellular senescence contributes to tissue and organismal aging, tumor suppression and progress, tissue repair and regeneration, and age-related diseas...
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