Anti-inflammatory and Analgesic Activities of Crotalaria pallida Aiton

• in CDCl3.[18]^1*

1. ¹ Fast‑atom bombardment mass spectrometry (FAB‑MS) (Joel SX 102/DA‑6000 mass spectrometer) was used to obtain the exact mass of turbiconol. Sterol sample (0.1 mg) in 100 μL HPLC grade methanol was used for the analysis. The quasimolecular ions (M + H) + spectrum were recorded at room temperature. The spectrum is obtained at resolution of 1000 amu. Mass spectra patterns were obtained with gas chromatography‑MS (GC‑MS) (Perkin Elmer, Clarus 680‑Clarus.
2. ² T) system. Sterol 1 mg was dissolved in 1 mL ethyl acetate and 1 µL was injected to GCMS equipped with nonpolar HP ultra‑double fused silica capillary column.[18] Novelty of the identified sterol was confirmed with the aid of SciFinder® . Cell culture Liver hepatocellular cells (HepG2) and human lung adenocarcinoma (A549) cell lines were purchased from the National Centre for Cell Science, Pune, India. Dulbecco’s modified Eagles media (DMEM/F2) was used and culture conditions were maintained at humidified 5% CO2 and 37°C of optimum temperature until a 70%– 80% confluence was reached. 10% Foetal Bovine Serum and antibiotics such as penicillin G (100 µL/mL), streptomycin (100 mg/mL), and amphotericin (250 ng/mL) were supplemented in culturing media. Trypsinization was performed with 0.5% (W/V) trypsin (0.25%, Invitrogen, USA) and ethylenediaminetetraacetic acid for 3 min at 37°C, only after attaining appropriate confluence. Appropriate cell suspensions obtained were used for further experimental procedures. Procedure To access the cytotoxic potential of turbiconol against lung and liver cancer, A549 and HepG2 human cell lines at a density of 2 × 105 cell/ mL seeded in a 96‑well microtiter plate was used. These cells were further incubated for 24 h at 37°C in a 5% CO2 atmosphere to ensure the complete cell adhesion. Cell adhesion to substrate was further verified using microscope. The plates were further used to access the antiproliferates mechanism and cell cytostatic activities. Cytotoxicity of turbiconol The changes in the cell viability were evaluated by thiazolyl blue tetrazolium bromide (3‑(4,5‑Dimethylthiazol‑2‑yl)‑2,5‑Diphenyltetrazolium Bromide [MTT] assay). Prior to turbiconol exposure to access cell viability well plates were seeded with 1000 trypsinized A549 and Hep G2 cell lines. After 24 h, these cells were treated with different concentration of turbiconol (6.5, 12.5, 25, 50, and 100 µg/mL) followed by 24 h incubation at 37°C in a 5% CO2 atmosphere. Negative controls (untreated cells) were included in all experimental runs. After turbiconol exposure for 24 h, media were carefully decanted from the plate and formazan product formed was dissolved in 200 µL dimethyl sulfoxide to evaluate the absorbance at 570 nm using microplate reader.[19] The data are reported as mean ± standard error (n test = 3). Morphological changes of cells after.

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