## 3 Results and Discussion  
Interactions of Oleanolic Acid and Polyphenols with Lipid Membranes--Steady-State Fluorescence Measurements  
Lipid membranes consist of a surface hydrophilic part and a hydrophobic core. Interactions between biologically active substances and these two regions can affect the biological properties of the membrane in different ways. Therefore, it is important to determine the localization of individual substances in different parts of the bilayer. In this study, fluorescent probes NBD-PE and DPH (diphenylhexatriene) were used to localize oleanolic acid and polyphenols in different parts of the bilayer. In the NBD-PE fluorescence probe, a moiety responsible for fluorescence emission is located on the surface of the lipid membrane. Consequently, it demonstrates interactions of compounds with the bilayer hydrophilic surface. Fluorescence quenching is caused by the interactions of the probe with water molecules. In turn, DPH is a hydrophobic molecule that localizes in the acyl chain region of a lipid membrane. In the presence of oleanolic acid, a decrease of NBD-PE fluorescence intensity at 534 nm was recorded at a triterpene concentration equal to 100 umol (Figure 2a). This means that OA at that concentration interacts with the bilayer and causes membrane structural changes leading to increased surface exposure to water molecules. Observed changes in DPH fluorescence emissions at 434 nm (Figure 3a), in the presence of oleanolic acid (at concentrations ranging from 5 to 100 umol), indicate the location of these molecules in the hydrophobic core of the bilayer. Interactions of OA with the lipid bilayer have already been observed by [PERSON] and coworkers [5]. It exhibits a moderate fluidity-modulating effect in liquid-crystalline liposomes made from dipalmitoyl phosphatidylcholine (DPPC) and a distinct condensing effect in liquid-crystalline and crystalline membranes. These effects are comparable to those observed in the presence of cholesterol. DPPC is membrane-substituted with saturated palmitic acid residues. In our study, natural phosphatidylcholine containing palmitic, oleic, linoleic and stearic acid residues was used. Such a membrane is liquid-crystalline at room temperature, i.e., under the conditions applied in this experiment. Subsequent experiments focused on the interaction of selected phenolic compounds (apigenin (AP), rutin (RT), resveratrol (RS) and ferulic acid (FA)) with phosphatidylcholine membranes containing 20 umol of OA. The increase of NBD-PE fluorescence intensity was recorded in the case of AP, RT and FA (Figure 2b,c,e). The observed changes are associated with the accumulation of polyphenols on the membrane surface, which subsequently hampers the quenching of probe fluorescence by water molecules. Such a mechanism of NBD-PE fluorescence decrease, resulting from the formation of hydrogen bonds with water molecules, was proposed by [PERSON] and collaborators [26]. The mildest inhibitory effect was observed in the case of ferulic acid, while the converse effect was noted for resveratrol (Figure 2d). In the presence of resveratrol, a decrease of about 40% in NBD-PE fluorescence intensity at 100 umol was found. Fluorescence-quenching was observed at RS concentrations higher than 20 umol, indicating changes in membrane structure leading to increased surface exposure to the aqueous environment. Further details concerning the interactions of phenolic compounds with the lipid membrane are revealed through measurements of the hydrophobic probe DPH fluorescence emission. At resveratrol concentrations of 5-20 umol, an increase in DPH emissions were recorded (Figure 3d), which indicates the embedding of polyphenol into the membrane's hydrophobic core. In  
Figure 1: MLP by Python generation procedure based on machine learning. turn, at RS concentrations ranging from 20 to 100 \(\upmu\)mol, a decrease of DPH fluorescence was found. This correlates well with NBD-PE emission-quenching at RS concentrations above 20 \(\upmu\)mol.