carnosol and sulforaphane

The Nrf2 signaling pathway is highly significant for redox homeostasis. Hence, nutrients and drugs activating Nrf2 can prevent oxidative stress-mediated medical conditions. After activation, Nrf2 accumulates in the cell nucleus; therefore, stimulation of Nrf2 by food components and drugs is usually monitored by measuring nuclear Nrf2 levels. The present study developed a targeted mass spectrometry method for the highly reliable quantification of nuclear Nrf2 levels. Three Nrf2-specific peptides were detected after enzymatic digestion of the nuclear fraction by the developed protocol for micro-liquid chromatography-tandem mass spectrometry in scheduled multiple reaction monitoring mode (microLC-MS/MS-sMRM). The method also identified nuclear Nrf2 unequivocally and specifically in the SDS-PAGE fraction of 100-150 kDa. Moreover, highly precise and linear relative quantification was achieved (mean relative standard deviation 8.3%; coefficient of determination 0.998). Incubation experiments in SH-SY5Y neuroblastoma cells revealed significantly up to 6-fold elevated nuclear Nrf2 levels after stimulation with 10 μM carnosol (rosemary), 10 μM sulforaphane (broccoli), or 20 μM cinnamaldehyde (cinnamon). Our results were in very good accordance with conventional Nrf2 western blotting and were highly correlated with the food components' effect on the expression levels of NAD(P)H dehydrogenase [quinone] 1 and thioredoxin reductase 1, two major Nrf2-regulated cytoprotective enzymes. The newly developed microLC-MS/MS-sMRM method shows broad applicability and can serve as a highly selective and reliable method to analyze Nrf2 activation. Graphical abstract ᅟ.

Topics: Abietanes; Acrolein; Anticarcinogenic Agents; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Nucleus; Functional Food; Humans; Isothiocyanates; NF-E2-Related Factor 2; Sulfoxides; Tandem Mass Spectrometry

A method was developed for targeted proteome analysis of the expression profile of a set of antioxidative enzymes in rat macrophages and applied to screen the antioxidative potential of several food components/foods.. Expression profiles of heme oxygenase 1, peroxiredoxin 1, thioredoxin reductase 1, glutathione reductase, glutathione-S transferase P1, and superoxide dismutase 1 were analyzed by nanoLC-MS/MS in selected scheduled reaction monitoring (sSRM) mode monitoring two to three peptides per protein and three transitions per peptide. Relative quantification was performed by metabolic labeling. The validated method was used to profile the activity of capsaicin, carnosol, diallyl trisulfide, maslinic acid, quercetin, sulforaphane, cinnamaldehyde and coffee extract to modulate the expression levels of antioxidative enzymes. Carnosol and sulforaphane most effectively induced protein expression, leading to upregulation of at least five out of the six antioxidative enzymes by a maximum factor of 22.80 ± 6.71 (heme oxygenase 1 by carnosol). Heme oxygenase 1 was most susceptible to nutritive modulation, whereas glutathione reductase expression rates were hardly affected.. Targeted mass proteome analysis allows comprehensive evaluation of antioxidative effects by food ingredients. Simultaneous expression analysis of a set of proteins provided valuable insights how various enzymes were differently affected by food components.

Topics: Abietanes; Animals; Antioxidants; Capsaicin; Cells, Cultured; Coffee; Food; Glutathione Reductase; Glutathione S-Transferase pi; Heme Oxygenase-1; Isothiocyanates; Macrophages; Proteome; Rats; Sulfoxides; Superoxide Dismutase; Thioredoxin Reductase 1