Anthrone method as described by Enneb, et al. [15]. The total carbohydrates content in the extracts was calculated employing a calibration curve of glucose and expressed as percentage of carbohydrates inside the extracts. The total protein content material was determined inside the extracts was obtained by Bradford colorimetric assay, as described by Enneb, Drine, Bagues, Triki, Boussora, Guasmi, Nagaz and Ferchichi [15]. The total protein content material was calculated utilizing a calibration curve of bovine serum albumin and expressed as percentage of protein within the extracts. As for the fat content material, it was determined utilizing Bligh and Dyer approach for total lipids analysis as described by Chua, et al. [16]. The total fat content material was determined gravimetrically and expressed as percentage of fat inside the extracts. Additionally, the moisture and ash quantification was also performed as described by Ferreira, et al. [17], determined gravimetrically and expressed as percentage inside the extracts. Lastly, the energy value of every extract was determined as described by Demoliner, et al. [18] using the Equation (two): Total power (kcal/100 g) = 4 ( g carbohydrates + g proteins) + 9 ( g Fat) two.six. Cell Culture, Cell Treatment and Determination of Citotoxicity Human Caco-2 cell line (TC7 clone) was kindly offered by Dr. Edith Brot-Laroche (UniversitPierre et Marie Curie-Paris six, UMR S 872, Les Cordeliers, France). The cells have been maintained inside a humidified atmosphere of 5 CO2 at 37 C. Cells have been grown in Dulbecco’s Modified Eagles medium (DMEM) (Gibco Invitrogen, Paisley, UK) supplemented with 20 fetal bovine serum (FBS), 1 non-essential amino acids, 1 penicillin (1000 U/mL), 1 streptomycin (1000 /mL) and 1 amphotericin (250 U/mL). The cells were passaged enzymatically with 0.25 trypsin-1 mM EDTA and sub-cultured on 25 cm2 plastic flasks at a density of 5 105 cells/cm2 . Culture medium was replaced just about every 2 days. Extract (two)Biomolecules 2021, 11,5 EP Inhibitor Source oftreatments had been added 24 h post-seeding for assays on Caco-2 undifferentiated cells (Garcia-Moreno et al., 2013); and 105 days post-seeding on differentiated Caco-2. Cell confluence (80 ) was confirmed by optical microscopy. The choice of those cells was associated with the possibility of functioning with the very same cell line in undifferentiated cancer cells and normal enterocytes. Extracts from avocado residues (peel, seed coat and seed) have been diluted in cell culture medium to the final concentration 1.2 mg/mL. A range of concentrations in the avocado extracts (62.five, 125, 250, 500, and 1000 /mL) was tested. These concentrations have been chosen based on prior operate, carried out by our analysis group, utilizing various plant extracts [191]. For cytotoxicity screening assays, the cells have been seeded in 96-well plates at a density of 4 103 cells/well. Culture medium was replaced with medium containing all distinct avocado extracts and cells had been incubated for 24, 48 or 72 h. Antiproliferative effect was measured together with the fluorometric cell viability Resazurin assay [22]. Resazurin stock option at a concentration of ten mg/mL was ready dissolving Resazurin powder in phosphate-buffered CB1 Antagonist Formulation saline (PBS) and this stock option was re-diluted 1:ten (with respect of total volume) in DMEM to receive the functioning answer. Immediately after removing culture medium from plates, one hundred of working option have been added to every single well and cells have been incubated at 37 C for 2 h. Finally, fluorescence was measured with excitation/emission at 544/590 nm applying a FLUOstar Omega (BMG Labtech).