2.16. SIRT1 Enzyme Activity Assay

SIRT1 enzymatic activity was assessed by using commercial kits (ab156065) from Abcam plc. (Cambridge, UK) in accordance with the manufacturer’s instructions. First, assay buffer (50 mM TRIS-HCl, pH 8.0, 137 mM sodium chloride, 2.7 mM potassium chloride, 1 mM magnesium chloride, 1 mg/mL bovine serum albumin), SIRT1 enzyme, and either solvent (DMF) or different concentrations of elaiophylin were mixed with the co-substrate (NAD) for 45 min. Thereafter, the stop/developing solutions, containing a mixture of a developer, were added to the microplate and incubated for 30 min at 25 °C. The deacetylated peptide reacts with the developer and releases a fluorophore. The fluorophores in both assays were analyzed at an excitation wavelength of 350 nm and an emission wavelength of 450 nm. The inhibitory percentage of the samples on the SIRT1 enzyme activity was calculated as the ratio of fluorescent intensity between samples and vehicle control [21].