Penicillin streptomycin solution 100

The CA was gifted by Ranbaxy (Mohali, India). Tween 20^®, ethyl oleate, eucalyptus oil, propylene glycol, polyethylene 400 (PEG 400), castor oil, dimethyl sulfoxide ≥99.5% (DMSO), thiazolyl blue tetrazolium bromide 98% (MTT) and methylcellulose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tween 80^®, soybean oil and olive oil were purchased from R&M Chemicals Ltd. (Essex, UK). Capmul MCM EP was obtained from the Abitec Corporation (Janesville, WI, USA). Peceol and Maisine 35-1 were obtained from Gattefosse (Saint-Priest, France). Cremophor EL^® was obtained from BASF (Ludwigshafen, Germany). The dialysis membrane (MWCO 12,000 g/mole) was obtained from Sigma-Aldrich Sdn Bhd, Petaling Jaya, Malaysia. The Caco-2 cell line was bought from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Dulbecco’s modified eagle’s medium (DMEM) was purchased from GE Healthcare Life Sciences (Logan, UT, USA). Trypsin 0.25% was purchased from GE Healthcare Life Sciences (Logan, UT, USA). The penicillin-streptomycin solution (100×) was obtained from Biowest (Nuaillé, France). Fetal bovine serum (FBS) and Dulbecco’s phosphate-buffered saline (DPBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The passive lysis buffer (5×) was purchased from Promega (Madison, WI, USA).

Cultures of human foreskin fibroblasts (HFFs) were maintained in Dulbecco’s modified Eagle medium (DMEM; Cat. No. D0819; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Cat. No. S181A; Biowest, Riverside, MO, USA) and 1% penicillin–streptomycin solution (×100; Cat. No. 168-23191; Wako, Osaka, Japan) at 37 °C and 5% CO₂. A cell viability assay was used to evaluate the cytotoxic action of Sm as described previously [18 (link)]. Briefly, a 100 μL cell suspension was seeded in a 96-well plate at a concentration of 1 × 10⁵ cells/mL in DMEM containing 10% FBS and incubated for 48 h at 37 °C and 5% CO₂. Next, 2-fold dilutions (total of eight concentrations) of test compounds in DMEM were added in quadruplicate to each well and incubated for an additional 72 h. After that, a Cell Counting Kit-8 (CCK-8; Cat. No. 343-07623; Dojindo, Kumamoto, Japan) was added, cells were incubated for an additional 3 h at 37 °C and 5% CO₂ and the absorbance was measured at 450 nm.