Chapter 4: Interaction of Plant Polyphenols with Liposomes

Abstract

Abstract The biological activities of plant polyphenols have been examined by various methods in vitro and in vivo for prediction of their ability to prevent human diseases. Their activities found in in vitro experiments with cultured mammalian cells should reflect the amount incorporated into the cells during incubation. Since no transporter specific to plant polyphenols has been found in mammalian cells, it is supposed that most polyphenols are incorporated into the cells by passive transport and the amount incorporated is related to the affinity of the polyphenol for the cell membranes. In general, measurements of the amount of a polyphenol incorporated into the cell membranes or the cells are difficult and inaccurate, because the amount is very low and some polyphenols are unstable and metabolized immediately after incorporation. We developed a method to measure the amount of polyphenols incorporated into the lipid bilayers of liposomes with a dense internal aqueous phase. The higher relative density of the liposomes than that of water enabled to separate the medium and the liposomes by ultracentrifugation for a short time after incubation. Consequently, the amount of a polyphenol incorporated into the separated liposomes was selectively measured. With this method, the affinities of various kinds of plant polyphenols, including phenyl propanoids, gallic acid esters, curcumin, flavonoids and isoflavones have been investigated. In particular, interaction of tea catechins with lipid bilayers has been investigated in detail. Epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECg) and epigallocatechin gallate (EGCg) are the major components of polyphenols in green tea infusions. EGCg is the most abundant among these compounds. The reported biological activities of EC and EGC were often lower than those of their corresponding gallic acid esters, i.e., ECg and EGCg. We clarified that EC and EGC had lower affinities for the lipid bilayers of liposomes than ECg and EGCg. This indicates that the interaction of tea catechins with the lipid bilayer partly governs their activities. In addition to the presence of the gallic acid ester, the number of the hydroxyl groups on the B-ring and the steric character of the C-ring affected the affinity of the catechins for the lipid bilayers. Furthermore, the external factors such as salt concentration in an aqueous medium of the liposome suspension, the electric charges of the lipid bilayers and the presence of EC governed the affinity of EGCg for the lipid bilayers. The interaction of EGCg with a model membrane was also examined by solid state 31 P and 2 H NMR. These NMR observations provide direct experimental evidence that EGCg molecule interacts with the lipid bilayers. Using liposomes with a dense internal aqueous phase, we also clarified that ECg and EGCg were located on the surface of the lipid bilayers and perturbed the membrane structure.