Molecular characterization of pasta enriched in antioxidants: investigation of their functional role by in vitro and in vivo approaches

1-Introduction: In agreement with the PhD thesis project described last year, this poster reports the main results of the first two activities concerning: (A1) Characterization and determination of the amount of starch, proteins and phenolics in different fractions of pigmented grains yielded by mechanical treatments. Monitoring the properties of the polysaccharides and proteins in the various pasta samples through novel methodologies (Bonomi et al., 2012). Studying the effect of extracts on glycaemia and on biomarkers of oxidative stress and inflammation by using inflamed Caco-2 line as a model. (A2) Producing pasta by incorporating pigmented grain flour in the nutritionally relevant amounts of fiber and anthocyanins, determining the nutritional/functional properties of the developed food products. 2-Materials and Methods: Pigmented grains were de-branned and milled in a lab-scale milling system. Extraction was conducted with ethanol and water (65:35 v/v) containing Hcl (%0.01). Analytical profiling of the phenolics in bran components/fractions separated by physical techniques was carried out by HPLC method. Free radical scavenging activity and the antioxidant capacity was measured by DPPH and FRAP method respectively. To study the anti-inflammatory effects we used a convenient way which relies on a reporter gene system in which Caco-2 cells are transfected with a vector (pNiFty2-Luc) which that enables facile monitoring of inflammatory state. The reporter gene encodes for a luciferase-labeled product, and is under control of a native promoter of the transcription regulator NF- B, which has a key role in the inflammatory response. In this system, the light emission (bioluminescence) is directly connected to the activation or repression of the promoter (Huang, Kim et al. 2010). Cells were supplemented with in vitro at different concentration of anthocyanin rich extracts in the presence/absence of interleukin (IL)-1ß, an important pro-inflammatory cytokine working as an activator of NF-B expression. Monitoring bioluminescence allows us for relatively easy estimation of the role of anthocyanins and/or metabolites with respect to the cellular response to stimulation. The α-glucosidase inhibitory activity was determined to measure antidiabetic effect of the pigmented grains extracts. 3-Results and discussion 3-1: Analytical profiling of the phenolics in the various fractions separated by physical techniques indicated that the outermost fraction obtained through debranning of purple wheat represents a very rich (and convenient) source of phenolics (and fiber, as assessed by independent measurements). The major anthocyanins found are cyanidin-3-o-glucoside and Petunidin-3-O- glucoside. DPPH and FRAP showed a relevant antioxidant activity with the anthocyanins amount in different fractions. The ACN-rich fraction from purple wheat decreased the immune response of Caco-2 cells in a dose-responsive manner, and wheat phenolics were apparently more effective than Cyanidines, that reportedly possess a strong anti-inflammatory activity (Yu and Ahmedna 2013).3-2: Enriched pasta were produced from different fractions of wheat flour and purple wheat bran, with fiber content in excess of 10%, and a content in anthocyanins in the 100 mg/kg range.Our results indicate that the phenolics-rich fraction from purple wheat at the same concentration to Cyanidine appears to be more effective in impairing the expression of NF-κB in transformed cells. Finding cyanidin-3-o-glucoside and Petunidin-3-O- glucoside as the main anthocyanin in purple wheat is in parallel with studding by Chen (Chen, Müller et al. 2013). These bioactive-rich fractions were incorporated in making pasta as staple food for human consumption to study its effectiveness on reducing the inflammation and hyperglycemia (Tsuda 2012, Cassidy, Mukamal et al. 2013). Although the bioavailability of anthocyanins and the amount of metabolites formed in the gastrointestinal tract, in order to introduce the desired anthocyanin efficacy should be considered during the following year of this PhD project, and also further studies will address the bioavailability of anthocyanins in pasta and the amount of metabolites they form or release in the gastrointestinal tract through studding their effects on the local microflora, and of the gut microflora interactions.