Intestinal pig epithelial cells (IPEC) J2 cells were used and grown for 8 days to confluency in Corning™ 6 well Transwell™ multi-well plates with permeable polycarbonate membrane inserts, using Dulbecco’s Modified Eagle (DME) medium and 5% fetal calf serum [32 (link),33 (link)]. The density of IPEC J2 cells was ∼2 × 106 cells/well initially and was grown to confluence. The plates had a polyester membrane thickness of 10 μm, and 0.4 μm pore size. The cells were washed with Dulbecco phosphate-buffered saline. One milliliter of saline was present in the bottom wells at all times. Twenty milligrams of curcuminoids, THC, or TurmiZn complex were solubilized separately in 10 mL saline with 5.0% dimethylsulfoxide (DMSO), and 1 mL of the respective solutions was placed in the top of six replicate wells per treatment, and incubated for 2 h at 37 °C. Optical density (OD) at 220 nm and 425 nm was then measured in the top and bottom wells by ultraviolet/visible (UV/Vis) spectrophotometry (Figure 1 A,B). The percent net average absorbance was calculated by dividing the bottom well absorbance by the total absorbances of the upper and bottom wells combined. High-performance liquid chromatography (HPLC) analysis was conducted on the upper and bottom wells at 280 nm on pooled samples (Figure 2 A). HPLC analysis was carried out with a Waters 2965 HPLC coupled with a photodiode array (PDA) detector (model 2996) along with Empower software. Column: Phenomenex Luna C18 250 × 4.6 mm, 5 µM. Isocratic: 50:50 acetonitrile: 2% acetic acid in water. Flow rate 1 mL/min for 20 min. The extraction solvent for analytes was acetone. The column temperature was 25 °C. PDA wavelengths 425 nm and 220 nm were used. The mean values ± standard error are reported.
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