Materials
Ligularia fischeri (LF) was collected at Inje-gun, Gangwon-do, Korea. RAW264.7 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). Low glucose (1000 mg/ml) Dulbeco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin cocktail were purchased from WELLGENE (Daegu, Korea). Lipopolysaccharide (LPS, Escherichia coli O55:B5), 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT), and 3-O-caffeoylquinic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Griess reagent was obtained from Promega (Madison, WI, USA). Nitric oxide (NO) and prostaglandin E2 (PGE2) assay kit were purchased from R&D System (Minnesota, USA). Reverse transcription and polymerase chain reaction premixes were from Applied Biosystems (Carlsbad, CA, USA).
Sample preparation
L. fischeri was collected at Inje-gun, Gangwon-do, Korea. LF was cooked in our laboratory, after cleaning and washing with water and cutting into small pieces. The LF was divided to provide 3 samples for fresh, blanching and pan-frying the rest was subjected to different cooking methods.
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(1)
Blanching in a stainless steel vessel: Washed LF (200 g) was added to water (2L) and blanched for 3 min.
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(2)
Pan-frying: Washed LF (200 g) was placed in a frying pan with oil and stirred for 3 min.
LF extracts were obtained using 70% ethanol with sonication (POWERSONIC 420, 700 W, 50/60 Hz, Hwanshin Tech.) for 40 min twice. Then the extracts of LF were filtered, evaporated (ELISA EVAPORATOR NVC-2000, SB-1000, DPE-1210, CA-1112, ELISA, Japan), and freeze-dried (FD5510, IlShin Lab Co., Ltd., Korea) to make powder samples. All samples were diluted to a 10 mg/mL concentration and used for the anti-inflammatory sample.
HPLC analysis
Reverse-phase high performance liquid chromatography (HPLC) was conducted using a Dianex u-300 system (Milford, MA, USA) that consists of Ultimate 3000 pumps, autosampler, and UV detector. The Chromeleon chromatographic system was employed to analyze the HPLC data. Chromatographic separation was accomplished using an Atlantis dC18 reverse phase column (Waters, 4.6 × 150 mm, 5 μm) and the elution was monitored at 300 nm. For separation, solvents A (Acetonitriles, ACN) and B (0.02% aqueous phosphoric acid, v/v) were used. The gradient program used was as follows: initial 0-6min, linear change from A-B (13:87, v/v) to A-B (15: 85, v/v); then held for 3 min; 9–17 min, linear change from A-B (15:85, v/v) to A-B (19:81, v/v); 17–28 min, linear change from A-B (17: 83, v/v) to A-B (28: 72, v/v); and then held for 9 min. The flow rate was 0.6 mL/min and an aliquot of 10 μL was injected.
Cell culture
Murine RAW264.7 macrophages were cultured in DMEM medium containing 10% FBS, peniciln, and streptomycin in a 5% CO2 humidified incubator at 37°C. RAW 264.7 cells were grown in 48-well plates at a density of approximately 5 × 104 cells per well.
Cell viability
Cells were treated with different concentrations (10, 50, 100 μg/mL) of LF for 24 hr. After that, the cells were incubated with MTT reagent, which was added to the culture medium at a final concentration of 0.5 mg/mL, for 4 hr in a 5% CO2 humidified incubator at 37°C. The resultant dark blue crystals were dissolved using dimethyl sulfoxide (DMSO) and absorbance values were measured at 540 nm.
Measurement of nitric oxide (NO) production
The production of NO was determined by measuring the accumulated level of nitrite, an indicator of NO in the supernatant. The RAW 264.7 cells were pretreated with or without LF for 1 hr. After LPS (1 μg/mL) was added to the cultured medium for 24 hr, nitrate levels were measured in cell culture supernatants according to the Griess reaction (1% sulfanilamide, 0.1% N-[naphthyl] ethylenediamine dihydrochloride, and 5% phosphoric acid) at room temperature for 10 min. Absorbance of the mixture at 550 nm was measured in a micro-plate reader (SpectraMax M2, Molecular Devices, USA). Nitrate concentration was calculated by comparison with a nitrite standard curve.
Measurement of prostaglandin E2 (PGE2) production
RAW 264.7 cells were seeded in 48-well plates (5 × 104 cells per wells) and incubated for 24 hr. The cells were treated with LF or a vehicle in the presence of LPS (1 μg/μL) for an additional 24 hr. PGE2 production in the cell supernatant was evaluated by PGE2 Parameter Assay kit following the manufacturer’s instructions.
Real-time quantitative polymerase chain reaction (RT-PCR)
RAW 264.7 cells were cultured in 6-well plates (5 × 105 cells per well) for 24 hr. The cells were treated with LF or a vehicle in the presence of LPS (1 μg/μL) for an additional 24 hr. Total RNA was isolated from the cells using RNAeasy kit (Qiagen) and then total RNA reverse-transcribed using a High Capacity RNA-to-cDNA™ Kit (Applied Biosystems) in order to produce cDNAs. Quantitative RT-PCR was performed in a Power SYBR® Green Master Mix (Applied Biosystems) under a STEPONE PLUS (Applied Biosystems), and the results were analyzed with the Stepone Software VER. 2.1 supplied with the machine. The housekeeping gene β-actin was used as an internal standard to quantify the levels of iNOS and COX2 mRNA. Parameters of RT-PCR reaction were 95°C for 5 min for one cycle, then 95°C for 15 sec, 61°C for 30 sec, and 72°C for 30 sec for 40 cycles. The fluorescence signal was detected at the end of each cycle. The primers used in the experiment were iNOS, forward: 5'- CCC TTC CGA AGT TTC TGG CAG CAG C -3', reverse: 5'- GGC TGT CAG AGC CTC GTG GCT TTG G -3'; COX2, forward: 5'- TCT CCA ACC TCT CCT ACT AC -3', reverse: 5'- GCA CGT AGT CTT CGA TCA CT -3'; and β-actin, forward: 5'- CCG TCT TCC CCT CCA TCG T -3', reverse: 5'- ATC GTC CCA GTT GGT TAC AAT GC -3'.
Statistical analysis
All experiments were repeated three times. All data are expressed as mean values standard deviation (SD). Statistical evaluations were made by ANOVA followed a Tukey’s HSD multiple comparison test. A value of p < 0.05 was considered significant.
