The aerial parts of Crytotaenia japonica Hassk. were collected in the Medicinal Herb Garden of Kyung Hee Univeristy (Yongin) in May 2009. A voucher sample specimen (CJ-01) was deposited in the laboratory of Herbology, College of Oriental Medicine, Kyung Hee University. The dried plant was boiled three times in 100% methanol for 2 h. The extract was filtered, concentrated in vacuo, and dried with a lyophilizer. The yield of the extract was approximately 24.7%. The powdered extract was dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) and sterilized by passing through a 0.22 μm syringe filter. A maximum of DMSO used for in vitro studies was 0.1%.
Gas chromatography / mass spectrometery
One mg of CJ methanol extract dissolved in 0.01 ml of DMSO was examined by gas chromatography coupled with mass spectrometer (Perkin Elmer Clarus 600T). A DB-5MS capillary column (30m x 0.25mm, film thickness 0.25μm) was used for the separation of constituents. The column temperatures were programmed from 50°C hold in initial 3 min to 140°C hold in 8.5 min, and then 310°C hold in 35 min. A constant flow rate of 1.0 ml/min was applied by using helium as the carrier gas. The electron energy for the mass selective detector was 70 eV. The temperature of the ion source was set at 255°C. Mass selective detector was used in SCAN mode over a mass scan range at m/z 50 to 600.
BALB/c mice (male, 8 weeks of age) were purchased from the Korean branch of Taconic, Samtaco (Osan, Korea), kept in a temperature-and humidity-controlled, pathogen-free animal facility at Kyung Hee University and provided standard mouse chow and water ad libitum. The mice were maintained in accordance with the Guide for the Care and Use of Laboratory Animals issued by the United States National Research Council (1996), and the protocol was approved by the Kyung Hee University Institutional Animal Care and Use Committee.
Isolation of peritoneal macrophages
Mice were injected intraperitoneally with 2 ml of sterile thioglycollate medium (BD, Sparks, MD, USA). Three days later, macrophages were collected by peritoneal lavage with cold Dulbecco’s modified Eagle’s medium (DMEM). Cells were resuspended in DMEM with 10% fetal bovine serum and incubated for 2 h in a humidified atmosphere of 5% CO2 at 37°C. Non-adherent cells were removed and the resulting adherent cell population consisted of 95% macrophages, as determined by morphology and non-specific esterase staining.
Cells were seeded at 4x104/ 0.1 ml in 96-well plates and stimulated for 24 h at increasing concentrations of CJ methanol extract. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopnehyl)-2H-tetrazolium (MTS) (Promega, Madison, WI, USA). Optical density was read at 490 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Measurement of nitrites
Cells were seeded at 2x106/ 2.0 ml in 6-well plates and primed for 2 h with 0.5 ng/ml of IFN-γ (BD Pharmingen, San Diego, CA, USA) before addition of LPS and CJ methanol extract. At 18 h after LPS stimulation, supernatant and cell pellets were used for subsequent assays. 50 μl medium was incubated with an equal volume of Griess reagent (Sigma) for 15 min at room temperature. The absorbance at 550 nm was measured with the microplate reader.
Supernatants or sera were appropriately diluted and the levels of cytokines were measured by ELISA according to the manufacturer’s protocol (BD Pharmingen).
Analysis of signaling molecules
Cells were seeded at 3x106/ 2.0 ml in 6-well plates and pre-treated for 1 h with CJ methanol extract and then stimulated with LPS for additional 15 min or 3 h. For the measurement of phospho-STAT1, cells were primed with IFN-γ.
Total proteins were prepared by resuspending the cells in lysis buffer (50 mM Tris–HCl, pH 7.5; 150 mM NaCl; 1mM EDTA; 20mM NaF; 0.5% NP-40; and 1% Triton X-100) containing a phosphatase inhibitor (Sigma) and a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Protein concentration was determined using the Bradford assay. Cell extracts were run on an 8% or 10% sodiumdodecyl sulfate-polyacrylamide gel and were transferred to polyvinylidene fluoride. The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h and then incubated overnight at 4°C incubated with IκBα, β-tubulin (Santa Cruz Biotechnology, CA, USA), iNOS (BD Pharmingen), phospho-IκBα, phospho-JNK, JNK, phospho-p38, p38, phospho-ERK1/2, ERK1/2, phospho-STAT1, or STAT1 (Cell Signaling Technology, CA, USA) diluted 1/1000 in 5% skim milk in TBST. The blots were washed with TBST and incubated for 1 h with anti-rabbit or anti-mouse HRP-conjugated antibody (diluted 1:5000 in 5% skim milk in TBST). Immunoreactive bands were developed using an enhanced chemiluminescence system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
In vivo experiment
CJ methanol extract dissolved in water ( 25, 100, and 400 mg/kg) was orally given for 1 week. On day 7, intraperitoneal injection of LPS (1.3 mg/kg) was performed and 1 h later mice were anesthetized with ether and blood was obtained by cardiac puncture.
Statistical differences among the means of multiple groups were determined by using one way ANOVA followed by Dunnet’s post hoc test. The difference between the two means was assessed using non-paired student’s t test. Calculations were carried out using SPSS version 12. P values less than 0.05 were considered significant.