Alkaline phosphatase activity

The effect on osteoblast-like cell differentiation was assessed by evaluating the alkaline phosphatase (ALP) activity of MG-63 cells cultured for six days in osteogenic medium [28] and then treated with olive oil phenolic extracts at doses of 10^-6M for 48 h. ALP activity was quantified using a colorimetric assay (Diagnostic kit 104-LL, Sigma, St. Louis, MO, USA) following Sandrini et al [29]. This assay measures the conversion by ALP enzyme of the colorless substrate p-nitrophenyl phosphate to the yellow product p-nitrophenol, with the color change rate corresponding to the amount of enzyme present in the solution. Standard solutions of p-nitrophenol (0–250 μM) were prepared from dilutions of a 1,000 μM stock solution and assayed in parallel. Cells were seeded into 24-well plates at 1×10⁴ cells/mL per well and cultured in the osteogenic medium under standard conditions for 7 days. The culture medium was then replaced by a fresh medium, and cells were treated with 10⁻⁶ M of the olive oil phenolic extracts under study, followed by their culture for 48 h under standard conditions. Untreated cells were used as control group. Finally, the cells were lysed with 0.1% (v/v) Triton X-100 at 37°C. Samples were then centrifuged at 1,500 rpm, and the supernatants were stored at −70°C until their use. ALP activity was determined by using p-nitrophenol phosphate as substrate, adding the cell lysate solution (50 μL) to 50 μL of ALP substrate (Sigma, St Louis, MO, USA) and incubating the resulting solution at 37°C for 45 min in darkness. The enzymatic reaction was stopped by adding 50 μL of 0.1 M NaOH, and absorbance was then measured at 405 nm with a spectrophotometer (Biotek EL×800). The total protein content was estimated with a protein assay kit from Bio-Rad Laboratories (Nazareth-Eke, Belgium) using Bradford’s method. All samples were run in triplicate, and specific ALP activity was expressed in U/mg cellular protein.