## 3 Results and Discussion
Results presented in Figure 1B-D clearly indicate the role of the activity of all extract components, the food matrix and the digestion stage in the generation of LPO activity. In the case of DE and AE, synergism was found, whereas pure chemicals (principal compounds of WF and GCB) and BE components exhibited a nearly additive interaction. Thus, drawing inferences concerning biological activity based only on knowledge of the activity of chemically pure compounds can lead to erroneous conclusions. Compounds derived from GCB and WF acted also as effective LOX inhibitors. As expected, the highest level of activity was determined for GCB extracts. Importantly, LOX inhibitors from GCB and WF were potentially bioaccessible and bioavailable in vitro (Figure 2B). Interestingly, the activity of pure chlorogenic and ferulic acids was quite similar (18.56 and 22.36 \(\upmu\)g/mL, respectively). This fact indicates the role of the food matrix in causing biological activity. Additionally, the isobole curve for ferulic/chlorogenic acids has a concave shape which indicates their synergistic action as LOX inhibitors (Figure 2A). Combination index (CI) analysis confirms this observation, the CI from pure chemical patterns is about 0.5, which confirms synergism. Also, hydrophilic (BE) and potentially bioaccessible(DE) compounds derived from GCB and WF demonstrate synergistic interaction (Figure 2C); however, the strongest synergism (CI = 0.40) was found for extracts after simulated digestion (Figure 2D). Most importantly, moderate antagonism was found in the case of potentially bioavailable compounds (AE)--the isobole has a convex shape, and the CI value (1.22) indicates moderate antagonism (Figure 2B,D). Lipoxygenases are enzymes containing non-heme iron and requiring catalytic activation. This activation process involves transformation of non-active iron in ferrous state to iron in ferric state, and this is accomplished by lipid hydroperoxide oxidation [4]. Several independent studies now support the correlation between 5-LOX expression and cancer cell viability, proliferation, cell migration, invasion through extracellular matrix destruction, metastasis, and activation of anti-apoptotic signaling cascades [26]. The abilities of plant extracts and pure chemicals to inhibit LOX activity are well known and extensively studied [27; 28]. While much is known about the interactions between active compounds and the food matrix [29; 30; 31], an often-overlooked problem is the interaction between the active compounds themselves. In this study, we found nearly additive interactions between CGA and FA (as LPO inhibitors) and synergism (as LOX inhibitors). Interestingly, taking into account chelating power, antagonism was found [25]. This fact may also confirm that Fe\({}^{2+}\)-chelating power is not the most critical factor to enable phenolics to inhibit lipids peroxidation. Figure 2: Lipoxygenase inhibition (LOXi) potential of green coffee beans and wholemeal wheat flour components in comparison with pure ferulic and chlorogenic acids. (**A**) Isobole curve for chlorogenic and ferulic acids, (**B**) LOXi potential of buffer extractable (BE), bioaccessible (DE), and bioavailable (AE) green coffee beans and wholemeal wheat flour compounds and pure ferulic and chlorogenic acids, (**C**) isobole curve for BE, DE, and AE green coffee beans and wholemeal wheat flour compounds, (**D**) estimation of strength of interactions of buffer extractable (BE), bioaccessible (DE), and bioavailable (AE) green coffee beans and wholemeal wheat flour compounds and pure ferulic and chlorogenic acids. An interesting issue is the question of the mechanisms of action of the studied extracts and their potential changes during digestion and absorption in vitro; thus, the next stage of our study was to determine the mechanism of action of LOX inhibitors in the light of their potential bioaccessibility and bioavailability. Both ferulic and chlorogenic acids acted as competitive LOX inhibitors (Figure 3A).