Wrl 68

Hepatoblastoma cell lines (huH6 and HepG2), normal liver cells (WRL68), and human bone marrow mesenchymal stem cells (BMSCs) were bought from ATCC (Manassas, VA, USA). Hepatoblastoma cells and WRL68 cells were maintained in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS and 1% streptomycin and penicillin (Thermo Fisher Scientific) in an incubator (37°C, 5% CO₂). BMSCs were maintained in α-MEM (Gibco) with 10% FBS (Corning) and 1% streptomycin and penicillin. The cells were maintained under the following conditions: 5% CO₂ and 95% humidity.

RIN-5F (rat pancreatic β-cell line) and WRL 68 (human hepatic cell line) (ATCC; Manassas, VA, USA) were maintained in RPMI-1640 and DMEM medium, respectively. 10% of fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin) were supplemented to the media, then it was incubated under an atmosphere of 5% CO₂ and 95% humidified air at 37 °C. The medium was replaced twice weekly until confluent cell monolayer was formed and observed under an inverted microscope.

Monolayer breast carcinoma cell lines (MCF-7), Human hepatocellular carcinoma cells (HepG2), arising retinal pigment epithelium (ARPE19) and hepatic human cell line (WRL 68) were obtained from ATCC (USA) and were separately cultured in growth media containing DMEM media, 10% heat inactivated foetal bovine serum (FBS), 100 U/mL penicillin, 100 U/mL streptomycin and 2 mM glutamine. All cell cultures were maintained at standard conditions at 37°C with 5% CO₂ humidity.

Human hepatocellular carcinoma cells (HepG2, SK-HEP-1, Chang Liver and WRL 68) were purchased from ATCC and maintained in our in house repository at National Centre for Cell Science, Pune, India. All the cells were grown in Dulbecco modified eagles medium (DMEM) either in normoglycemic glucose, 5.5 mM (NG) or in high glucose, 25 mM (HG), depending upon the experiments and supplemented with 10% heat inactivated fetal bovine serum (Hyclone, UT, USA), penicillin (100 U/ml) and streptomycin 100 μg/ml (Invitrogen Life Technologies, CA, USA), at 37 °C in an incubator in the presence of 5% CO₂ (Thermo Scientific, NC, USA).

All reagents and solvents were purchased from Merck (Darmstadt, Germany) and Acros Organics (New Jersey, US) and used without further purification. Flash chromatography was performed using silica gel with 200–300 mesh produced by Merck. All reactions and processes of flash chromatography were monitored by the TLC method using silica gel plates with fluorescence F254 and iodine visualization. The melting points were determined with Stuart melting point apparatus SMP30 and uncorrected. Fourier-transformed infrared absorption spectra of both solid and liquid samples were analysed with NICOLET 6700 FT-IR using diamond with ATR.
Microanalyses were performed on Flash Elemental Analyzer 110 series. The ¹H NMR and ¹³C NMR spectra were recorded on a Joel- 400 spectrometer at 400 MHz at 125 MHz using CDCl₃ and DMSO-d₆ as solvents and TMS as internal standard. Coupling constants (J) are expressed in hertz (Hz). Chemical shifts (δ) are given in parts per million (ppm). All target compounds have purity over 95%.
All ATCC bacterial strains (BAA-1556, BAA-1688, ATCC 6358, ATCC 35556, ATCC 25923 and ATCC 33591) and the three mammalian cell lines (WRL-68, Vero CCL-81 and BALB/3T3) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The additional 37 S. aureus clinical isolates were obtained from three local Hospitals in Peninsula Malaysia.