Reagents Dimethylsulfoxide (DMSO), LPS, Griess’s reagent, was obtained from the Sigma Co (St. Louis, MO). All other reagents were of the first grade. Preparation of Extract Mangifera indica (MI) stem bark was collected from market. The stem bark extract of M. indica was prepared by decoction for 1 h in accordance to the method of (Garrido et al. 2004) as described earlier (Dhananjaya et al., 2011). Cell culture RAW264.7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 U/ml of penicillin and 100 µg/ml of streptomycin, and 5 % FBS. Cells were grown at 37oC and 5% CO2 in humidified air. Cells were incubated with. MI at different concentrations (25, 50,100, 200 μg/ml) or positive chemical and stimulated with LPS 0.1μg/ml for 18hrs. Cell Viability test A cell proliferation assay was also performed to exclude the possibility that the observed NO inhibition was false positive due to the cytotoxic effects. Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described previously (Hong et al., 2003). All experiments for the measurement of nitric oxide inhibition were conducted three times, each time with three independent observations and the results were averaged. Measurement of nitrite In order to determine the concentration of NO, nitrite (NO2 - ) was measured using the Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2% phosphoric acid), as described previously (Hong et al., 2003). After preincubation of the RAW 264.7 cells (1X106 cells/ml) for 24 h, the cells were incubated with 25, 50, 100 or 200 μg/ml of water extracts of MI with LPS (0.1 μg/ml) for 18 h. One-hundred μl of supernatant from each well of the cell culture plates were transferred into 96-well micro plates, and the supernatant was then mixed with equal volume of Griess reagent at room temperature. The absorbance at 550 nm was determined in a Spectramax 250 micro plate reader. The concentrations of nitrite were calculated from regression analysis using serial dilutions of sodium nitrite as a standard. The percentage inhibition was calculated based on the ability of extracts to inhibit NO formation by cells compared with the control (cells in media without extracts containing triggering agents and DMSO), which was considered as 0% inhibition.
Extraction of total RNA and Semiquantitative RT-PCR Total RNA from the RAW264.7 cells were prepared by adding Easy blue Reagent (iNtRON Biotechnology Co. Korea), according to the manufacturer’s instructions. The total RNA solution was stored at -70 until used. Semi quantitative RT reactions were carried out using RT premix (Bioneer Co. Korea). Briefly, total RNA (2 μg) were incubated with oligo-dT18 at 70? for 5 minutes and cooled on ice for 3 minutes, and for 90 minutes after the addition of RT premix at 42.5. The reactions were terminated at 95? for 5 minutes for the inactivation of reverse transcriptase. The PCR reaction was further conducted using the PCR premix (Bioneer Co. Korea) with the appropriate sense and antisense primers for Glyseraldehyde-3-phosphate dehydrogenas (GAPDH, sense primer, 5’-CAC TCA CGG CAA ATT CAA CGG C3’; antisense primer, 5’-CCT TGG CAG CAC CAG TGG ATG CAG G-3’), iNOS (sense primer, 5’- CCC TTC CGA AGT TTC TGG CAG CAG C-3’; antisense primer, 5’- GGC TGT CAG AGC CTC GTG GCT TTG G-3’), COX-2 (sense primer, 5’- CAC TAC ATC CTG ACC CAC TT -3’, antisense primer, 5’- ATG CTC CTG CTT GAG TAT GT - 3’), interleukin (IL)- - CAG GAT GAG GAC ATG AGC ACC-3’; antisense primer, 5’- CTC TGC AGA CTC AAA CTC CAC-3’), interleukin (IL)-6 (sense primer, 5’-GTA CTC CAG AAG ACC AGA GG-3’; antisense primer, 5’-TGC TGG TGA CAA CCA CGG CC3’), tumor necrosis factor (TNF)- - TTG ACC TCA GCG CTG AGT TG -3’; antisense primer, 5’- CCT GTA GCC CAC GTC GTA GC-3’), and GM-CSF (sense primer, 5’- AGG ATG TGG CTG CAG AAT TTA CTT TTC -3’; antisense primer, 5’- TCA TTT TTG GAC TGG TTT TTT GCA TTC -3’) under incubation conditions (a 45-second denaturation time at 94', an annealing time of 45 seconds at 55 to 60', an extension time of 45 seconds at 72', and final extension of 10 minutes at 72' at the end of the cycles. The PCR products were separated in 1% agarose using electrophoresis of BioRad Co. The relative intensities were calculated using Eagle eyes image analysis software (Stratagene Co. La Jolla). The resulting densities of the iNOS, COX-2, IL-1, TNF-α, and GM-CSF bands were expressed relative to the corresponding densities of the GAPDH bands from the same RNA sample. GAPDH, a housekeeping gene, was used as RNA internal standard. Statistical analysis: Data were analyzed by a one-way analysis of variance, followed by a post-hoc Dunnett's test. All data are presented as means ± S.E.M.