5 % w/v sodium hydroxide, and 0.1 % v/v NaOCl) were added to each well and the increasing absorbance at 625 nm was measured after 20 min,

using a microplate reader (Molecular Device, USA). The percentage inhibition was calculated from the formula 100 − (OD test well/OD control) × 100. Thiourea was used as the standard inhibitor. In order to calculate IC50 values, different concentrations of synthesized compounds and standard were assayed at the same reaction conditions (Weatherburn, 1967). The obtained results are presented in Table 2. Table 2 Inhibitory activities of the synthesized compounds against Jack Bean urease Compound % Inhibition ± S.D. IC50 ± S.D. Thiourea 100 ± 0.1 54.56 ± 4.17 2 -a –b 3 11 ± 3.3 – 4a N.s. – 4b N.s. – 4d – – 4e 1 ± 0.2 – 4f – – 5 – – 6 3 ± 3.0 – 7 N.s. – 8 7 ± 3.1 – 9 7 ± 3.0 – 10 4 ± 1 – 12 56 ± 4 VX-680 – 14 – – 15 100 ± 1.5 4.67 ± 0.53 17 100 ± 2.1

45.37 ± 0.78 18 – – 19a – – 19b 47 ± 0.1 – 19c – – 20 N.s. – N.s. Not soluble aNo inhibition bNot determined Anti-lipase activity assay The inhibitory effects of those compounds were evaluated against porcine pancreatic lipase (PPL) (15 ng mL−1). Lipase activity assay was done according to Verger et al., (Woods et al., 2003). Microtiter plates were coated with purified tung oil TAGs. Compounds were mixed with PPL 1:2 (v/v) and incubated for 30 min. The microtiter plates containing purified tung oil, lipase solution, and assay buffer (10 mM selleck chemicals llc Tris–HCl buffer, pH 8.0, containing 150 mM NaCl, 6 mM CaCl2, 1 mM EDTA, and 3 mg mL−1 β-cyclodextrin) were recorded continuously for 40 min against the buffer alone by using microplate reader (SpectraMax M5, Molecular Devices) at 272 nm. The inhibitory activity

of those compounds and Orlistat, a positive control against pancreatic lipase, were measured at concentration of 6.25, 2.08, and 1.04 μg mL−1. Residual activities were calculated by comparing to control without inhibitor (T+). The assays were done in triplicate. The IC50 value was determined as the concentration of compound that give 50 % inhibition of maximal activity. The results are presented in Table 3. Table 3 Porcine pancreatic lipase inhibitory activity of synthesized compounds Compound no. % Inhibition 2 – 3 – 5 – 6 16 7 33 8 22 9 20 10 – 11 – 12 68 13 63 14 75 15 73 16 6 17 – 18 1 19a ADP ribosylation factor – 19b – 19c – 20 33 Orlistat 99 DMSO control – Positive control – All compounds were screened at concentration of 6.25 μg mL−1 Acknowledgments This Project was supported by Scientific and Technological Research Council of Turkey (TUBITAK, Project No: 107T333) and Karadeniz Technical University, BAP, Turkey (Ref. No. 8623) and is gratefully acknowledged. Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.