The whole plant of Taraxacum coreanum Nakai was collected from the Botanical Garden of Wonkwang University (Iksan, Korea) in May 2014. This species was identified by Dr Kyu-Kwan Chang, and the voucher specimen (WK-2014-028) was deposited in Wonkwang University. Each fresh aerial and root part (50 g) was soaked in 500 ml ethanol and left for 7 days at room temperature. Following filtration with filter paper, the solvent was dried using rotary evaporator to yield 7.5 g of aerial ethanolic extract (TCAE) and 12.8 g of root ethanolic extract (TCRE).

Chromatography was performed using a HPLC instrument of the YL-9100 series (YoungLin Instrument Co., Ltd., Anyang, Korea). In all experiments, a Capcell Pak C18 column (4.6×250 mm, 5 µm; Shiseido Co., Ltd, Tokyo, Japan) was used as the stationary phase, and the injection volume was 20 µl. Samples containing 2 mg/ml of TCAE or TCRE were prepared. The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B) in a gradient system: 0–50 min linearly changed 10 to 50% B, 50–55 min linearly changed 50 to 100% B, 55–60 min remained at 100% B. The detection wavelength was adjusted to 254 nm, and the flow rate was 0.7 ml/min.

All cell culture reagents were obtained from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Tin protoporphyrin (SnPP) and cobalt protoporphyrin IX (CoPP) were obtained from Frontier Scientific, Inc. (Logan, UT, USA). Caffeic acid, chlorogenic acid, ferulic acid, and all other chemicals, unless indicated otherwise, were obtained from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany).

Mouse hippocampal HT22 cells were obtained from Professor Hyun Park (Wonkwang University). Cell culture and MTT assay were conducted as described previously (12). Briefly, a total of 2×104 cells/well were seeded in 96-well plates. They were pre-treated with the indicated concentration of TCAE or TCRE for 3 h, and this was followed by treatment with 5 mM glutamate. For measurement of cell viability, cells were maintained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at a final concentration of 0.5 mg/ml for 4 h, and the formazan formed was dissolved in acidic 2-propanol.

HT22 cells were harvested and pelleted by centrifugation at 200 × g for 3 min. Subsequently, the cells were rinsed with PBS and lysed using radioimmunoprecipitation assay (RIPA) lysis buffer containing 25 mmol/l Tris-HCl buffer (pH 7.6), 150 mmol/l NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS for 15 min at 4°C, and then underwent centrifugation at 15,000 × g for 10 min at 4°C. The protein concentration was determined using Bradford Assay Reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A total of 30 µg protein samples were resolved using SDS-polyacrylamide gel electrophoresis and electrophoretically transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk, washed with TBST buffer, and then incubated with the following primary antibodies: Anti-HO-1 (catalog no. sc-10789; 1:1,000), anti-Nrf2, (catalog no. sc-722; 1:1,000) anti-Lamin B, (catalog no. sc-6216; 1:1,000), anti-Actin (catalog no. sc-1616; 1:1,000) all from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). They were then incubated with horseradish peroxidase-conjugated goat (catalog no. ap106p; 1:1,000) and rabbit (catalog no. ap132p; 1:1,000) secondary antibodies, obtained from EMD Millipore (Billerica, MA, USA), followed by ECL detection. Primary and secondary antibodies were diluted with 3% skimmed milk in TBST buffer. The bands were visualized with enhanced chemiluminescence (GE Healthcare Life Sciences, Chalfont, UK) and quantified by densitometry (Image J, National Institutes of Health, USA). Nuclear and cytoplasmic extracts of cells were prepared using NE-PER reagents, as per the manufacturer's instructions (Thermo Fisher Scientific, Inc.).

Determination of HO activity occurred as previously described by Motterlini et al (13). Briefly, the HT22 cells were scraped off the dish, and centrifuged (1,000 × g for 10 min at 4°C). The pellet of HT22 cell was suspended in MgCl₂ phosphate buffer (20 mM, pH 7.4), frozen at −70°C, and finally sonicated on ice prior to centrifugation at 18,000 × g for 10 min at 4°C. The supernatant was added to a nicotinamide adenine dinucleotide phosphate (NADPH)-generating system containing 0.8 mM NADPH, 2 mM glucose-6-phosphate, 0.2 U glucose-6-phosphate-_L-dehydrogenase, and 2 mg protein of rat liver cytosol prepared from the 15,000 × g supernatant fraction as a source of biliverdin reductase, potassium phosphate buffer (100 mM, pH 7.4), and hemin (10 µM) in a final volume of 200 µl. The reaction was conducted for 1 h at 37°C in the dark and terminated by addition of 1 ml chloroform. The extracted bilirubin was calculated by the difference in absorption between wavelengths of 464 and 530 nm using a quartz cuvette (extinction coefficient, 40 mM-1 cm-1 for bilirubin).

HT22 Cells were homogenized (1:20, w:v) in PER-Mammalian Protein Extraction buffer (Pierce; Thermo Fisher Scientific, Inc.) including freshly added 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail I (EMD Millipore). The cytosolic fraction of the cell was prepared by centrifugation at 15,000 × g for 10 min at 4°C. Cytoplasmic and nuclear extracts of the cells were prepared using NE-PER cytoplasmic and nuclear extraction reagents (Pierce; Thermo Fisher Scientific Inc.), respectively. These methods were conducted as previously described (12).

Data were expressed as the mean ± standard deviation of at least 3 independent experiments. To compare 3 or more groups, one-way analysis of variance followed by the Newman-Keuls post hoc test was used. Statistical analysis was performed using GraphPad Prism software version 3.03 (GraphPad Software Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Three phenolic compounds, ferulic acid, caffeic acid and chlorogenic acid, have been isolated from plants of the Taraxacum genus (14,15). Therefore, TCAE and TCRE were analyzed by HPLC in order to assess the presence of these compounds. As demonstrated in Fig. 1, the peaks of ferulic acid and caffeic acid appeared clearly in the HPLC chromatograms of both TCAE (Fig. 1A) and TCRE (Fig. 1B), however, chlorogenic acid was only detected in TCRE (Fig. 1B).

To investigate the protective effects of TCAE and TCRE against glutamate-induced cytotoxicity, HT22 cell viability was measured by MTT assay. Non-cytotoxic effects were obtained with concentrations of up to 400 µg/ml for both TCAE and TCRE (data not shown). The viability of glutamate-treated HT22 cells (5 mM for 24 h) was tested following treatment with 0, 50, 100, 200 and 400 µg/ml of each extract (Fig. 2). The results demonstrated that both TCAE and TCRE significantly restored the cell viability following glutamate-mediated damage in a concentration-dependent manner compared with cells treated with glutamate only (Fig. 2). Trolox, a well-known antioxidant, was used as a positive control in this assay and was confirmed to exhibit a significant protective effect (Fig. 2). The present results suggested that both aerial and root ethanolic extracts of T. coreanum could protect HT22 cells against oxidative stress.

The effect of TCAE and TCRE on HO-1 expression was examined in HT22 cells. When cells were treated with 50, 100, 200 or 400 µg/ml TCAE or TCRE for 12 h, both extracts visibly increased the expression of HO-1 protein in a concentration-dependent manner, compared with untreated cells (Fig. 3A). In agreement with the concentration-dependent expression of HO-1, both TCAE and TCRE treatments also significantly increased HO-1 activity in HT22 cells compared with untreated cells (Fig. 3B). CoPP, used as a positive control, exhibited a prominent induction of both HO-1 protein expression and activity (Fig. 3).

The hypothesis that the protective effect of TCAE and TCRE in HT22 cells arose from HO-1 expression was further tested. Pretreatment of cells with SnPP, a competitive inhibitor of HO-1, significantly reduced the cytoprotective effects of TCAE and TCRE on glutamate-induced cell damage (P<0.05; Fig. 4). The present data, therefore, suggested that the neuroprotective effects of TCAE and TCRE against oxidative stress are a result of their ability to induce HO-1 expression.

In order to examine whether Nrf2 is involved in the extract-mediated HO-1 induction, the effect on Nrf2 nuclear translocation in HT22 cells treated with TCAE and TCRE was tested (Fig. 5). HT22 cells were incubated with 400 µg/ml TCAE (Fig. 5A) or TCRE (Fig. 5B) for 30, 60 and 90 min, then Nrf2 protein levels in the nuclear and cytosolic fractions were examined by western blot analysis. The nuclear extracts of both TCAE and TCRE-treated cells exhibited a time-dependent increase in Nrf2 levels compared with untreated control, while Nrf2 levels decreased in the cytoplasmic fraction of HT22 cells (Fig. 5).