Plasma creatine kinase activity was determined using the CK-UV Kit (Bioclin, Brazil). Activity was expressed in units/L, with one unit corresponding to the production of 1 μmol of NADH per min at 30 °C. Negative controls received 50 μL of 1% DMSO-PBS alone. The control group for each sesquiterpene lactone compound received an intramuscular PD-1 antibody inhibitor injection of 50 μL of a solution containing

just 20 μg of each sesquiterpene lactone derivative compound, dissolved in 1% DMSO in PBS (pH 7.2) ( Souza et al., 2008 and Da Silva et al., 2008a). The measurement of the enzymatic activity using the micellar substrate, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol (HPGP), was performed through the microtiter plate assay. Each sesquiterpene lactone derivative compound was tested in final concentrations of 0.0, 1.0, 2.0, 3.0 and 4.0 μM. Seven wells of a 96-well microtiter plate were used for each assay, resulting in six measurement repetitions of the enzymatic activity for each final concentration of the inhibitor. Thus, for each assay with different concentrations of the inhibitor, 100 μL of solution A in assay buffer (27 μM bovine serum albumin, 50 mM KCl, 1 mM CaCl2, 50 mM Tris– HCl, pH 8.0) was added to seven wells, followed by the addition of a volume of lactone find more derivative compound (the volume used was according to the assay concentration,

0.0–4.0 μM) dissolved in DMSO. For control reactions, the same volume of DMSO was used Morin Hydrate alone. Solution B had the same composition as that of Solution A, but contained PLA2 (0.5 μg/mL) and was delivered in 100 μL volumes to seven wells, except for the first well. Instead of Solution B, an additional

100 μL-portion of Solution A was added to the first of the seven wells in the assay. Solution B was prepared immediately before each set of assays to avoid loss of enzymatic activity. Quickly, after the addition of Solution B, the assay was initiated by the addition of 0.5–50 μL of Solution C to the seven wells (53 mM HPGP vesicles in assay buffer), with a repeating pipette. The final concentration of HPGP varied from 0.125 to 10 mM. The final volume of the assay was 265 μL and, when necessary, the wells received an extra volume of solution A in order to complete this volume. The fluorescence (excitation = 342 nm, emission = 395 nm) was read with a microtiter plate spectrophotometer (Fluorocount, Packard Instruments). Control reactions without enzyme or inhibitor were run for all assays and the initial velocity was calculated from the initial slope of fluorescence versus time, for each concentration of the substrate used. The significance of differences between groups was evaluated using the Student’s t-test. A P-value <0.05 was considered to be significant ( Da Silva et al., 2008b). The structures of each sesquiterpene lactone derivative compound were submitted to ab initio quantum calculations. In order to select the best conformations, the HyperChem 7.51 software was utilized.