Andrographolide

T84 cells (70 – 80% confluent) were incubated with serum free DMEM:F12K containing one third the concentration of antimicrobials (3,330 U/ml penicillin, 3,330 μg/ml streptomycin, 8.3 μg/ml amphotericin B) for 3 h. Media was replaced with DMEM:F12K medium containing 2% FBS in the presence of andrographolide (Sigma Aldrich, St. Louis, MO) at IC₅₀ for 24 and 48 h. ER stress was blocked in some experiments by adding 4-PBA (Sigma Aldrich) to the cells 30 min prior to andrographolide treatment. In some experiments, cells were treated with 1 μg/ml tunicamycin (TM) in parallel as a positive control for ER stress and apoptosis. [28 (link)]

Andrographolide was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and dissolved in DMSO (Merck, Darmstadt, Germany) to obtain a stock solution of 100 mM. Andrographolide was further diluted in culture medium to the appropriate final concentrations prior to use.

Quantitative analysis of andrographolide of A. paniculata was carried out as per the protocol of Masaenah et al. [49 (link)] with minor modification using HPLC (Thermo ScientificTM DionexTM UltiMate 3000 UHPLC, Waltham, MA, USA). Chromatographic separation was performed using XBridge^® (Waters, Milford, MA, USA) C18 column (5 µm, 4.6 mm × 250 mm). The mobile phase consisted of a mixture of methanol and deionized water (50:50) and a 1 mL/min flow rate. The injection volume was 10 μL. The temperature of the column was controlled at 25 °C, and samples were detected using a PD-M20A photodiode array detector. The andrographolide (Merck Chemical, Saint-Quentin Fallavier, France) was identified by comparing the retention time of samples with reliable standard chromatographic peaks at 254 nm, and the andrographolide content was expressed as µg/mL.