## 3 Results and Discussion
In our experiment, the phosphatidylcholine membrane was in the liquid-crystalline phase at room temperature, indicating that our results are similar to those of [PERSON] and co-workers. [PERSON] and co-workers [30] found that apigenin at the concentration 0.625-10 \(\upmu\)mol decreases the fluidity of phosphatidylcholine membranes (acyl chains 18:1/16:0 ratio equal to 1) containing 20 mol% of cholesterol. In the case of our study, a bilayer was incorporated with 20 \(\upmu\)mol of OA, which may affect its structure, similarly to cholesterol. The hardening effect of apigenin has also been confirmed for plasma membranes of L929 cells [31]. Figure 4: Fluorescence anisotropy of NBD-PE probe in lipid membrane in the presence of oleanolic acid (a), and oleanolic acid mixed with apigenin (b), rutin (c), resveratrol (d) and ferulic acid (e). Influence of Oleanolic Acid and Polyphenols on Phosphatidylcholine Liposomes Hydrodynamic Diameter--Dynamic Light Scattering (DLS) Measurements  
DLS measurements reveal the hydrodynamic diameter of liposomes. This parameter can be influenced by various factors, such as the aggregation or the appearance of substances that change the hydrophilic-hydrophobic balance. In the presence of OA, the decrease of a membrane's hydrodynamic diameter from about 245 nm (PC membrane without OA) to 175 nm (100 umol OA) was observed (Figure 6a). [PERSON] and co-workers [32] found that sterols structurally similar to OA increase the size of liposomes made from  
Figure 5: Fluorescence anisotropy of DPH probe in lipid membrane in the presence of oleanolic acid (**a**), and oleanolic acid mixed with apigenin (**b**), rutin (**c**), resveratrol (**d**) and ferulic acid (**e**). egg yolk phosphatidylcholine. In their research, the mean vesicle size was 82 nm, and had increased to about 110 nm, 175 nm and 325 nm after addition of 40% of cholesterol, \(\beta\)-sitosterol and stigmasterol, respectively. However, in up to 20% of sterols no significant differences were observed. The authors highlight that the planar and non-bulky structure of the cholesterol molecule is less likely than \(\beta\)-sitosterol and stigmasterol to cause steric hindrance, which results in the formation of smaller liposomes. In our experiment, at 100 \(\upmu\)mol OA the concentration of this compound was 57% of PC content. Thus, with a comparable ratio of OA to membrane, the obtained effect was a reduced hydrodynamic diameter of liposomes, quite different from that observed in the presence of cholesterol, \(\beta\)-sitosterol and stigmasterol. From NBD-PE fluorescence measurements, it is evident that 100 \(\upmu\)mol OA located in the hydrophobic core of the membrane causes structural changes leading to increased exposure of the bilayer surface to the aqueous environment. Hence, it can be assumed that OA causes the rearrangement of phospholipid packing in the bilayer, which leads to a change in the membrane curvature and the formation of liposomes of smaller size. In subsequent experiments, the impacts of phenolic compounds on phosphatidylcholine-choline membranes enriched with 20 \(\upmu\)mol of OA were investigated. In the case of apigenin (Figure 6b), the decrease of the liposomes' hydrodynamic diameter from about 275 nm to about 140 nm at 100 \(\upmu\)mol of AP was found. [PERSON] and co-workers [33,34] prepared distearoyl phosphatidylcholine (DSPC) liposomes containing apigenin (DSPC:apignin ratio--2.5:1). They found, in two experiments, that the hydrodynamic diameter of membranes loaded with AP was slightly higher (103.2-104.3 nm) in comparison with empty DSPC (91.84-94.07 nm). The increased diameter of membranes loaded with AP suggests the formation of hydrogen bonds between flavonoids and the hydrophilic part of the bilayer [35,36], as confirmed in studies with dipalmitoyl phosphatidylcholine (DPPC) membranes.