Spectramax m2

The total phenolic content for each berry sample (CEs, ACFs, ACEs) was determined using the Folin–Ciocalteau method and expressed as gallic acid equivalents (GAEs) as previously reported [12 (link)]. Briefly, the berry samples (10–20 mg/mL) were diluted 1:100 with methanol/H₂O (1:1, v/v). 100 μL of each sample was incubated with 50 μL of Folin–Ciocalteau reagent for 5 min at room temperature (25 °C). 150 μL Na₂CO₃ and 250 μL H₂O were added to each sample and incubated at 40 °C in the dark for 30 min. Samples were then cooled on ice to room temperature and centrifuged. The absorbance was determined at 756 nm on a Spectramax M2 plate reader operated by SoftmaxPro v.4.6 software (Molecular Devices, Sunnyvale, CA, USA). The total anthocyanins content was performed using the pH differential method (expressed as cyanidin-3-O-glucoside equivalents) as previously reported [57 (link)]. Briefly, each berry sample was made to a stock solution (20 mg/mL). Two aliquots (1.0 mL) of the stock solution were placed into 25 mL volumetric flasks which were then filled up to mark using pre-prepared buffers of pH 1.0 or pH 4.5, respectively. The absorbance of these buffer solutions were recorded at 510 and 700 nm, respectively, using a Spectramax M2 plate reader (Molecular Devices, Sunnyvale, CA, USA).

For deoxycholic acid, ileal content (50 mg) was homogenized in 2 mL of ice-cold PBS using an Omni TH^TM homogenizer (Omni International, Kennesaw, GA, USA). The homogenate was incubated at 4 °C for 20 min, then transferred to a microcentrifuge tube, and centrifuged at 12,000× g for 20 min. The supernatant was transferred to a fresh microcentrifuge tube and stored at −20 °C for deoxycholic acid analysis. Deoxycholic acid was analyzed using a commercially available ELISA kit (Deoxycholic acid; LSBio, Seattle, WA, USA) following the manufacturer’s instructions. The homogenates were either diluted 1:10, 1:20, or 1:40.
For cholic acid, plasma samples were diluted 1:10, and the cholic acid level was determined using a commercially available ELISA kit (cholic acid; Cell Biolabs, San Diego, CA, USA) following the manufacturer’s instructions.
The concentrations of both deoxycholic acid and cholic acid were measured at 450 nm absorbance on the plate reader (SpectraMax M2, Molecular Devices, San Jose, CA, USA), and the results were calculated from a standard curve determined for each plate using the SpectraMax M2 software.