Chemopreventive potential of oat derived phytochemicals. The role of naturally isolated and chemically synthesized avenanthramides.

The present research project was designed to evaluate the main parameters which affect the nutrient level during the formulation of oat-based food products. The focus was on avenanthramides (AVNs), a group of more than 30 phenol compounds found exclusively in oats. The three main AVNs (2c, 2p and 2f) were isolated by us, as a mixture (n-MIX), from oat sprouts, as well as chemically synthesized, in order to test their health protective effects, namely antioxidant, anti-proliferative and anti-inflammatory activities. First, we described the morphology of oat caryopsis, in order to localize nutrients within its layers, with particular attention to phenol compounds. Conventional optical microscopy coupled with specific staining techniques, as well as Environmental Scanning Electron Microscopy-Energy Dispersive Spectroscopy enabled us to identify macronutrients, whereas autofluorescence allowed us to observe phenol compounds in the outer layers of oat caryopsis. Second, we evaluated the effects of genotype/environment interactions and oat processing, on phenol concentration. Our results indicated that genotype was the main determinant on AVN levels, as naked oats showed about three-fold higher values than dehulled oats. Nevertheless, also the interaction soil x genotype was relevant on AVN content. Regarding the mechanical processing, the greatest loss of phenol antioxidants was observed after dehulling, as AVNs were also found in hulls; milling reduced bound polyphenols only, due to their covalent bound with the fibrous matrix. Third, we evaluated the increase of AVN levels during malting, in order to obtain functional products, namely oat-based cookies, which underwent to simulated digestion process to evaluate the recovery of antioxidants. Minor AVNs, namely 2s, 2pd, 2fd, in addition to the three main forms, were identified by us in malted oats and in their derived cookies. Total AVNs showed a ten-fold increase after five days of malting, probably due to enzymatically catalyzed de novo biosynthesis. Cooking did not affect the AVN content of our bakery products, whereas the in vitro digestion provided a relative bioaccessibility, ranging from 10 to 55%, depending on the individual AVN form. Finally, we evaluated the main biological effects exerted by the n-MIX or synthetic AVNs s-2c, s-2p, s-2f. Among the three synthetic forms, the s-2c showed the highest antioxidant capacity, detected by three different methods (DPPH, ABTS, ORAC), due to the presence of two hydroxyl groups in the molecule. The study of their anti-proliferative effect was carried out on one normal cell line (NCTC 2544) and three different cancer cell lines (CaCo-2, HepG2, Hep3B). On NCTC 2544 no cytotoxic effect was observed in the range 0-120 µM, whereas on cancer cell lines the n-MIX and the s-2c showed the greatest citotoxicity. The anti-tumor activity was exploited by AVNs by targeting the extrinsic apoptotic pathway, as demonstrated by the activation of caspases 3, 8, 2. The strong pro-apoptotic effect of AVNs was linked to: high membrane permeability, as ascertained by their cellular antioxidant capacity; downregulation of the pro-survival factors, like the hypoxia inducible factor (HIF1A) and the vascular endothelial growth factor (VEGFA), as well as the cyclooxigenase (COX-2). The overall conclusion is that malting can be a good method to increase AVN levels in oats. The choice of the cultivar, as well as the mechanical processing and storage conditions are key factors to ensure the maintenance of high levels of AVNs in the final oat-based functional products.