Nordihydroguaiaretic acid ndga

The composition of PSS (in mM) was 119 NaCl, 4.7 KCl, 1.2 KH₂PO4, 25 NaHCO₃, 1.2 MgSO₄, 11.1 glucose, and 1.6 CaCl₂. The composition of 60 mM KCl solution (in mM) was 59 NaCl, 60 KCl, 1.2 KH₂PO4, 25 NaHCO₃, 1.2 MgSO₄, 11.1 glucose, and 1.6 CaCl_2. All salts were purchased from Sigma Aldrich (Schnelldorf, Germany). XE991 was purchased from Tocris (Bristol, UK). 5-HT, phenylephrine (PE), 4-aminopyridine (4-AP), nordihydroguaiaretic acid (NDGA), 17-octadecynoic acid (ODYA), 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA) were purchased from Sigma Aldrich (Schnelldorf, Germany). PAME were purchased from Cayman Chemical (Ann Arbor, Michigan, USA).
Data were analyzed by Prism version 5.0 (GraphPad Software, La Jolla, California, USA) and were shown as mean ± SD or mean ± SEM. Paired, unpaired Student's t-tests or one-way ANOVA were used as appropriate. In Figures 1C, 2C, statistical significance was determined by two-way ANOVA or repeated-measures two-way ANOVA, followed by Bonferroni post-hoc test, and using Prism 6 software. A value of P < 0.05 was considered statistically significant; n represents the number of arteries tested.

In vitro inhibition of soybean LOX was followed the previously described method^{8 (link)}. The tested compounds were dissolved in DMSO as 10 mM stock solutions. 10 μL were incubated at room temperature with sodium linoleate (100 mM) and 0.2 mL of enzyme solution (1/9 × 10⁻⁴ w/v in saline) in buffer pH 9 (tris) at room temperature (final volume 1 mL). The conversion of sodium linoleate to 13-hydroperoxylinoleic acid at 234 nm was recorded. The results were compared with the appropriate standard inhibitor NDGA (IC₅₀ = 0.45 μM). Several concentrations were used for the determination of IC50 values. The results are given in Table 1 expressed as IC₅₀ values or % inhibition at 100 μM and compared with the appropriate standard inhibitor. Nordihydroguaiaretic acid (NDGA) (Aldrich Chemical Co. Milwaukee, WI, (USA), was used as a positive control. To determine the IC₅₀ values, several dilutions of compounds were used. Blank determination served as a negative control.

Several assays were used to evaluate in vitro antioxidant activity of nitrones, such as the inhibition of lipid peroxidation (LP), induced by AAPH in the presence of atmospheric oxygen, competition of the tested compounds with DMSO, in terms of hydroxyl radical scavenging activity, and ABTS^+∙ decolorization assay. In vitro inhibition of soybean lipoxygenase (LOX) was used to determine anti-inflammatory activity. Reagents and materials: nordihydroguaiaretic acid (NDGA), Trolox, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), soybean LOX, and linoleic acid sodium salt were from Aldrich Chemical Co. Milwaukee, WI, (USA). Phosphate buffer (0.1 M, pH 7.4) was prepared by mixing an aqueous KH₂PO₄ solution (50 mL, 0.2 M), and an aqueous NaOH solution (78 mL, 0.1 M); pH (7.4) was adjusted by adding a solution of KH₂PO₄ or NaOH. A Lambda 20 (Perkin–Elmer-PharmaSpec 1700, Boston, MA, USA) UV–Vis double beam spectrophotometer was used for the assays.