Lipoxygenase Inhibitory Effects of Dibenzylbutane Lignans from the Seeds of Myristica fragrans (Nutmeg)

Experimental

General. Soybean lipoxygenase-1 (EC 1.13.11.12, Type 1), linoleic acid and Tween-20 were purchased from Sigma (Sigma Chemical Co, St. Louis, MO, USA). Compounds 1-7 were obtained from our previous work.7 NDGA (nordihydroguaiaretic acid) was purchased from Aldrich (Milwaukee, WI, USA). All reagent grade chemicals were purchased from Sigma (St. Louis, MO, USA).

Enzyme Assay. The enzyme assay was performed as previously reported21 with a slight modification. Briefly, 10 μL of an ethanolic inhibitor solution was mixed with 60 μL of 1 mM stock solution of linoleic acid and 2.925 mL of 0.1 M Tris-HCl buffer (pH 8.0) in a quartz cuvette. Then, 5 μL of a 0.1 M Tris-HCl buffer solution (pH 8.0) of lipoxygenase (1.02 μM) was added. The resultant solution was mixed well followed by reading at 234 nm for 5 min, which represents the formation of conjugated diene hydroperoxide (13-HPOD, ε = 25000 M−1 cm−1). A lag-period shown in the lipoxygenase reaction22 was excluded for the determination of initial rates. The stock solution of linoleic acid was prepared with methanol and Tris-HCl buffer at pH 8.0, then total methanol content in the final assay was adjusted below 1.5%. Two concentrations (20 and 40 μM) of linoleic acid were selected for Dixon plots. The assay was conducted in triplicate of separate experiments. The data analysis was performed by using Sigma Plot 2000 (SPSS Inc., Chicago, IL). The inhibitory concentration leading to 50% activity loss (IC50) was obtained by fitting experimental data to the logistic curve by the equation as follows:

Activity (%) = 100 [1/(1+([I]/IC50))]

Inhibition mode was analyzed by Enzyme Kinetics Module 1.0 (SPSS Inc.) equipped with Sigma Plot 2000.

Molecular Docking. All the simulations were performed on Linux workstations using InsightII 2005 software package (Accelrys, CA, USA). The crystal structure of soybean lipoxygenase-1 (PDB ID: 1YGE) determined at 1.4 Å resolution23 was used for the molecular docking studies. Hydrogens, charges and potentials were assigned using the CVFF force field and the protein was energy minimized. For the ligand preparation, DDGA (7) and MDGA (3) were sketched with the help of Builder module in InsightII 2005. Then the charges & potentials were assigned using CVFF forcefield and the minimization was done using the Discover module in InsightII 2005 with 1000 steps of steepest descents followed by 5000 steps of conjugate gradients. For molecular docking, the program GOLD (Genetic Optimisation for Ligand Docking, Cambridge Crystallographic Data Centre, UK)24 was employed to dock the DDGA (7) and MDGA (3) into the substrate binding site of soybean lipoxygenase. Active site radius of 10.0 Ǻ was defined from the Fe metal atom coordinated to H499, H504, H690, N694, I839 residues. The RMS deviation was considered within 1.5 Ǻ and the annealing parameter of van der vaals interaction was 4.0 Ǻ, hydrogen bond interaction was 2.5 Ǻ.