ReviewInfluence of membrane lipid composition on flavonoid–membrane interactions: Implications on their biological activity
Introduction
Flavonoids are a group of polyphenols ubiquitously distributed in vegetables, fruits, seeds, roots and stem either in the aglycone form or as glycone derivatives [1]. All flavonoids possess a typical structure comprising three cyclic carbon rings denoted as A, B and C. The substitutions in the ‘C’ ring are used to distinguish different flavonoid subgroups [1], [2]. Flavonoids possess numerous pharmacological activities such as anti-cancer, anti-diabetic, anti-atherosclerotic, anti-oxidant, anti-inflammatory properties, etc [1], [2]. They are also consumed as food supplements. The pharmacological activities of flavonoids have been primarily attributed to their ability to alter the membrane-mediated signaling pathways resulting in modification of cell membrane permeability and also to their binding with specific protein targets [3]. Fig. 1 shows the structure of flavonoids and their major subgroups.
It has now been recognized that plant-derived drugs have different molecular targets in the cells that include the cell membrane, intracellular compartments and DNA [4], [5], [6]. The gateway for any molecule to enter into the cell is the plasma membrane and hence membrane interactions of a molecule will have a major influence on their mechanisms of action [3], [7], [8], [9], [10], [11], [12]. Consequently, the biological activities of a molecule will be a manifestation of its mode of interaction with the membrane components as well as its localization and residence time in the cell membrane. As the lipid bilayer construct is a universal component of all membranes, interactions of molecules with the membrane lipid components can be termed as ‘non-specific’ interactions. Such interactions can also alter the functions of the membrane-associated proteins [13]. The membrane permeation of a molecule is chiefly associated with the nature and extent of its interaction with cellular membranes [8], [14]. In the case of flavonoids, the mode of membrane interaction has been mainly attributed to their lipophilicity and planar structure [15]. The number and position of hydroxyl groups influences the relative hydrophobicity of different flavonoids [3], [16], [17]. The presence of a 2,3 double bond in the B ring and absence of glycosidic substituent in the flavonoid structure tends to render it more planar [1]. Thus, it is evident that a strong correlation exists between the structure and biological activity of a flavonoid.
Another major parameter that can influence the biological activity of flavonoids is the fluidity of the membrane. Plasma membrane offers a diffusion barrier that protects the cell cytoplasmic contents from adverse interactions of molecules. However, it transforms into a highly permeable structure when subjected to oxidative stress leading to cell dysfunction and death [13], [18]. Oxidative stress-induced membrane damage due to lipid peroxidation has been implicated in various free radical-mediated diseases such as neuronal degeneration, atherosclerosis, cancer, rheumatoid arthritis, etc., all of which are associated with altered membrane fluidity [19]. Apart from free radicals, different exogenous compounds are also known to cause similar changes in the packing density of the membrane lipids on interaction with the plasma membrane [20]. Flavonoids have been reported to localize either in the hydrophobic core of the lipid bilayer or at the membrane interface leading to corresponding alterations in the membrane fluidity or rigidity [3], [7], [8]. The alteration of membrane fluidity by flavonoids can significantly influence the membrane-mediated cell signaling pathways. Thus, the structure of the flavonoids as well as their ability to alter the membrane fluidity are both important factors that influence the nature and magnitude of their biological activity. This review attempts to elaborate the mode of membrane interaction of flavonoids and their biological importance with respect to lipid composition and types of membranes. This will pave a way for understanding the pharmacological importance of membrane-mediated signaling cascades for developing novel therapeutic agents.
A scan of literature reveals that though the importance of flavonoid–membrane interactions has been duly recognized, widely contrasting reports exist on their mode of interactions. The reasons for such conflicting reports may arise due to a combination of many factors. One of the reasons for the observed variations in the results could be the widely varying concentration of the flavonoids employed in these studies [8]. The concentration of the flavonoid could determine the magnitude of molecular stress at the membrane surface and in the interior of the bilayer that would be reflected in the observed effects of the interaction. Yet another aspect that could contribute to the conflicting reports on the effects of flavonoid–membrane interactions is the type of membrane model used for the study. The most common membrane models employed for such studies are planar lipid bilayers and liposome vesicles [21], [22], [23], [24], [25]. The lipid composition employed for forming the membrane models will have a pronounced effect on the nature as well as extent of flavonoid–membrane interactions apart from influencing their localization within the lipid bilayer. The most common lipids that have been employed for forming membrane mimics are dipalmitoyl phosphatidyl choline (DPPC), distearoyl phosphatidyl choline (DSPC), palmitoyl oleoyl phosphatidyl choline (POPC) and cholesterol. Fig. 2 depicts the lipid membranes formed using these lipids.
A distinct feature in these membrane bilayers is the difference in their lipid-packing factor. Membranes formed from DPPC or DSPC are more compactly organized with fewer packing defects due to the strong hydrophobic associative forces that exist between the saturated fatty acyl chains of DPPC or DSPC. In contrast, membranes formed using phospholipids containing unsaturated acyl chains such as POPC possess larger number of packing defects due to the cis double bond in the unsaturated acyl chain that prevents close association of the neighboring acyl chains [26]. Such membranes are more fluid than those formed from DPPC or DSPC. Introduction of cholesterol in the membrane tends to rigidify it because of the presence of stiffer phenanthrene rings [26]. However, increasing the cholesterol content beyond a certain limit has been found to impart greater fluidity to the membrane bilayer. This has been attributed to the mismatch in the chain length of the phospholipid acyl chains and the cholesterol structure [27]. As a result, more voids are created in the bilayer, thereby transforming it into a highly permeable structure.
The cell membrane consists of different types of lipids along with proteins. The distribution and composition of the lipids depends upon the cell type. Among the different membrane lipids, phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidyl serine are the most predominant in the cellular membrane, whereas phosphatidylinositol is present in smaller quantities. The interactions of the membrane phospholipids with other molecules influence the fluidity and packing of the lipid membrane. The polymeric nature of lipid arrangements, physical properties such as cross sectional area, fluidity, electric charge and molecular weight play a major role in these interactions.
A variety of techniques have been employed to understand the mode of interaction of flavonoids with membranes. These include electrochemical methods, nuclear magnetic resonance spectroscopy (NMR), electron spin resonance spectroscopy (ESR), differential scanning calorimetry (DSC), and fluorescence anisotropy techniques [14], [21], [23], [28]. Each technique will provide insights into a particular facet of flavonoid–membrane interactions. A correlation of the data obtained from each technique is therefore necessary for complete understanding of the nuances involved in the interactions between flavonoids and membranes.
Section snippets
Strategies employed for elucidating flavonoid–membrane interactions
Many techniques have been reported to probe different aspects of the flavonoid–membrane interactions. These are elaborated in the following sections:
Differential scanning calorimetry
Phase transition behavior of lipid components in the presence of absence of flavonoids can serve to understand the alteration in membrane fluidity caused by these molecules using differential scanning calorimetry (DSC) [10], [16]. The interactions of flavonoids with the phospholipids constituting the bilayer may either serve to restrict acyl chain motion thereby bringing about a positive shift in the phase transition temperature or may induce packing defects that can reduce the phase transition
Liposome aggregation studies
To better understand the influence of flavonoid localization on membrane fluidity, turbidity measurements to determine the extent of liposome aggregation in the presence of flavonoids has been reported in literature [25]. Daidzein, an isoflavone was found to induce liposome aggregation in a dose-dependent manner that was attributed to a decrease in the hydration of the membrane surface by daidzein [25]. Different lipids were employed for the study and it was observed that negatively charged
Fluorescence anisotropy
The technique involves the use of a fluorescent probes and application of polarized fluorescence excitation light beam. Depolarization of emission light depends directly on tumbling motions of probe molecules within the bilayer [41]. The technique involves the use of a fluorescent probe whose emission characteristics in different directions vary depending on its tumbling motions within the bilayer [41]. The most commonly employed fluorescent lipid probe is diphenyl hexatrienyl (DPH)
NMR spectroscopy
Solid state NMR techniques have also been employed to confirm the membrane localization of flavonoids [23]. The presence of a molecule in the hydrophobic chain is expected to alter the magnetic environment around the proton and carbon nuclei in the immediate vicinity of the molecule, which will be reflected in the chemical shift values recorded in the 1H and 13C NMR spectrum respectively. Applicability of this technique in membrane fluidity studies is associated with the fact that the shape of
Electrophysiological methods
Among the different methods employed to understand the membrane localization and alteration in membrane fluidity, monitoring changes in the electrical behavior of black lipid membrane (also known as planar bilayer lipid membrane, BLM) is a simple and well known tool [14]. The amphipathic phospholipids self-assemble to form planar lipid bilayers spontaneously when introduced in an aperture bifurcating two aqueous compartments. This bilayer mimics the membrane bilayer architecture and presents a
Molecular dynamics simulation
The mode of interaction of flavonoids has also been investigated using molecular dynamics simulations. In silico studies have been extensively used for predicting potential drug targets and the nature of their interaction with pharmacophores from promising drug candidates [52]. Molecular dynamics simulates the motions of molecules and helps to predict the possible sites where molecule–molecule interactions are most likely to occur. Such studies have been employed to decipher protein-drug
Membrane interaction studies using RBC membrane models
The native cellular membrane comprises different types of proteins, which mediate membrane associated cell-signaling pathways apart from phospholipids and cholesterol [13], [56], [57], [58]. Cholesterol and membrane proteins along with the phospholipids mainly control the fluidity of the cell membrane. Therefore, the findings of the study employing a completely lipidic membrane model can be extended to a more realistic biological membrane by employing a model membrane system that comprises both
Biological significance of membrane interactions of flavonoids
Several reports have suggested a relation between membrane interactions and the biological activities of flavonoids [3], [6], [32], [35], [45], [77], [78]. It has been reported that the anti-tumor activity of some flavonoids is mediated by functional changes induced in several membrane-associated proteins. These include inhibition of Ca2+–Mg2+ ATPase, Na+–K+ ATPase, mitochondrial ATPase, cAMP- and cGMP-mediated phospho diesterase activities [18], [79], [80]. The extent of interaction of
Conclusion and future prospects
Membrane localization and alteration in membrane fluidity by flavonoids have emerged as key factors in determining their anti-cancer and anti-oxidant properties. A scan of literature reveals that change in membrane fluidity mainly depends on the composition of lipids and concentrations of flavonoids used in each study. Each technique employed to understand the membrane interaction of flavonoids provides specific insights about their localization or alteration in membrane fluidity. It is also
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
The authors wish to acknowledge financial support from the Department of Science & Technology (SR/SO/BB-35/2004) and SASTRA University for financial and infrastructural support.
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