Selenium (Se) is an essential trace nutrient for humans and animals owing to its role in redox regulation, thyroid hormone control factors, immunity, inflammatory reactions, brain activities, and carbohydrate regulation. It is also important to support muscle development, as well as for reproductive and cardiovascular well-being. Furthermore, sulfur is known to be a healing element, due to the remarkable function of specialized and secondary S-containing compounds. The scope of the current study was to determine the impact of Se and S enrichment on the secondary metabolite accumulation and antibacterial and NO inhibition activities in green and red leaf lettuce (V1 and V2, respectively). The plants were grown in a hydroponic system supplied with different S concentrations (S0: 0, S1: 1 mM and S2: 1.5 mM K2SO4) via the nutrient solution and foliar-applied varying levels of Se (0, 0.2 and 2.6 µM). Electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI-QTOF/MS) combined with ultra-performance liquid chromatography (UPLC) was used to identify the secondary metabolites in green and red lettuce. The results indicated that extracts of the biofortified lettuce were not cytotoxic to Vero kidney cells at the highest concentration tested of 1 mg/mL. The ESI/MS of the tentatively identified metabolites showed that the response values of 5-O-caffeoylquinic acid, cyanidin 3-O-galactoside, quercetin 3-O-(6''-acetyl-glucoside) and quercetin 3-O-malonylglucoside were induced synergistically under higher Se and S levels in red lettuce plants. The acetone extract of red lettuce had antibacterial activity against Pseudomonas aeruginosa, with a minimum inhibitory concentration (MIC) of 0.156 and 0.625 μg/mL under S2/Se1 and S2/Se2 treatments, respectively. As with antibacterial activity, the acetone extract of green (V1) lettuce treated with adequate (S1) and higher S (S2) under Se-limiting conditions showed the ability to inhibit nitric oxide (NO) release from macrophages. NO production by macrophages was inhibited by 50% at respective concentrations of 106.1 ± 2.4 and 101.0 ± 0.6 μg/mL with no toxic effect on the cells, in response to S1 and S2, respectively, under Se-deficient conditions (Se0). Furthermore, the red cultivar (V2) exhibited the same effect as the green cultivar (V1) regarding NO inhibition, with IC50 = 113.0 ± 4.2 μg/mL, in response to S1/Se2 treatments. Collectively, the promising NO inhibitory effect and antibacterial activity of red lettuce under the above-mentioned conditions might be attributed to the production of flavonoid glycosides and phenylpropanoic acid esters under the same condition. To the best of our knowledge, this is the first report to show the novel approach of the NO inhibitory effect of Se and S enrichment in food crops, as an indicator for the potential of Se and S as natural anti-inflammatory agents.
Keywords: NO; UPLC; antibacterial; cytotoxicity; lettuce; secondary metabolites; selenium; sulfur; synergistic interaction.