*Article* **Study of Resveratrol's Interaction with Planar Lipid Models: Insights into Its Location in Lipid Bilayers**

**Daniela Meleleo**

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari "Aldo Moro", via E. Orabona 4, 70126 Bari, Italy; danielaaddolorata.meleleo@uniba.it; Tel.: +39-080-5442775

**Abstract:** Resveratrol, a polyphenolic molecule found in edible fruits and vegetables, shows a wide range of beneficial effects on human health, including anti-microbial, anti-inflammatory, anti-cancer, and anti-aging properties. Due to its poor water solubility and high liposome-water partition coefficient, the biomembrane seems to be the main target of resveratrol, although the mode of interaction with membrane lipids and its location within the cell membrane are still unclear. In this study, using electrophysiological measurements, we study the interaction of resveratrol with planar lipid membranes (PLMs) of different composition. We found that resveratrol incorporates into palmitoyl-oleoyl-phosphatidylcholine (POPC) and POPC:Ch PLMs and forms conductive units unlike those found in dioleoyl-phosphatidylserine (DOPS):dioleoyl-phosphatidylethanolamine (DOPE) PLMs. The variation of the biophysical parameters of PLMs in the presence of resveratrol provides information on its location within a lipid double layer, thus contributing to an understanding of its mechanism of action.

**Keywords:** resveratrol; planar lipid membrane; cholesterol; channel-like event; capacitance

#### **1. Introduction** ′

Trans 3, 4 ′ , 5-trihydroxystilbene, also known as *Trans*-resveratrol (Figure 1), is found in edible fruits and vegetables, particularly in grapes, blueberries, blackberries, and peanuts.

**Figure 1.** Structure of *Trans*-resveratrol.

Resveratrol is also present in red wine, giving rise to the so-called "French paradox" by which, despite the fat-rich diets, mortality from coronary heart disease is lower in France than in other countries due to the inhabitants' moderate consumption of red wine [1].

Besides its cardio-protective effects, resveratrol has shown a wide range of beneficial effects on human health, including anti-microbial [2], anti-inflammatory [3,4], anti-cancer [5,6], and anti-aging [7,8] properties. Some studies have shown that this polyphenol has a preventive effect on Alzheimer's disease and dementia, due to its antiinflammatory properties [9–15].

Numerous studies have been carried out in order to gain an understanding of the mechanisms underlying the beneficial effects of resveratrol [16]. The results of studies carried out by Yashiro et al. [17] and Voloshyna et al. [18] show that its cardio-protective

**Citation:** Meleleo, D. Study of Resveratrol's Interaction with Planar Lipid Models: Insights into Its Location in Lipid Bilayers. *Membranes* **2021**, *11*, 132. https://doi.org/ 10.3390/membranes11020132

Academic Editor: Monika Naumowicz Received: 18 January 2021 Accepted: 10 February 2021 Published: 14 February 2021

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effect may be due to resveratrol's capacity to act as a modulator of lipoprotein metabolism. Indeed, resveratrol inhibits the oxidation of low-density lipoproteins and platelet aggregation, while increasing high-density lipoproteins [19]. It is known to be a scavenger of free radicals [20]. Several studies have shown that, when phospholipase A2 promotes the release of cytokines and NADPH oxidases that cause cell inflammation, resveratrol has proven to act as a good cell protective agent [21–24].

Although resveratrol's beneficial effects on human health have been accepted, its molecular mechanism remains unclear. The biomembrane seems to be the main target of resveratrol, although its mode of interaction with membrane lipids and its location within the cell membrane are subject to debate. Resveratrol's affinity for the lipid matrix is indicated by its poor water solubility (0.03 g/mL) and high partition coefficient (3.07 and 3.11 in 1,2-dipalmitoyl-*sn*-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-*sn*-glycero-3-phosphocholine (DSPC) liposomal membranes, respectively) [25].

Numerous studies have examined the interaction and location of resveratrol within the membrane utilizing different techniques and lipid models given that the cell membrane is a complex system. The results of these studies are not unique. Indeed, it has been either concluded that resveratrol is located in the hydrophilic headgroup region or, on the contrary, in the hydrophobic core.

The results of a study carried out by de Ghellinck and colleagues indicate that resveratrol accumulates between the lipid headgroups, causing conformational changes in the tilt angle of the lipid headgroups to a more upright orientation [26]. Incorporation and location of resveratrol in the hydrophilic headgroup region seem to depend on the lipid composition of the membrane model. The study carried out by Han and colleagues shows that, in 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) liposomes, resveratrol could penetrate into the lipid membrane, locating mostly in the headgroup region rather than in the deeper region of the lipid double layer. In DOPC/sphingomyelin/cholesterol or sphingomyelin/cholesterol liposomes, resveratrol associates with the liposome surface without penetrating into the headgroup region. This different behavior shown by resveratrol may be due to a different packing of the lipid acyl carbon chains. In DOPC liposomes, the acyl carbon chains are less packed than in liposomes containing sphingomyelin and cholesterol [27]. Similar results have also been obtained by Cardia and colleagues, who studied the interaction of resveratrol with soybean phosphatidylcholine (P90G) liposomes by <sup>1</sup>H NMR spectroscopy. Resveratrol is located in the region of the phosphocholine headgroup. Quantitative data on the incorporation of resveratrol at both the liposome preparation stage and by preformed liposomes suggest that the amount of resveratrol incorporated into P90G liposomes was about 20 mM, which corresponds to a 150-fold increase with respect to the solubility of resveratrol in water. These experimental data show that resveratrol diffuses through lipid bilayers [28]. Using liposomes made from saturated phosphatidylcholine (DPPC) and cholesterol (CHOL) or positively charged derivates of cholesterol (DC-CHOL), Bonechi et al. found that resveratrol associates with the surface of liposome containing CHOL, as found by Han et al. [27], while the polyphenolic compound inserts more deeply into cationic liposomes [29].

On the other hand, some authors state that resveratrol is located in the hydrophobic core. The results of a study carried out by Balanˇc and colleagues indicate that resveratrol is incorporated in the inner part of the liposome membrane [30]. In agreement with the results obtained by Balanˇc are those presented by Fabris and colleagues who, using phosphatidylcholine liposomes of various chain lengths, show that the hydroxyl groups of resveratrol are located in the lipid region of the bilayers close to the double bonds of polyunsaturated fatty acids and that the longer the acyl chains, the more stable and less perturbable the bilayers [25]. Similar results have also been obtained by Brittes and colleagues [1]. In addition, Neves and colleagues show that resveratrol induces phase separation and formation of liquid-ordered domains in liposomes made of egg L-α phosphatidylcholine, cholesterol and sphingomyelin. Besides, this polyphenol stabilizes the membrane bilayers of the

liposome locating in a deeper region of the membrane and adopting a vertical position in the nonpolar region of the membrane, with two hydroxyl groups in the interfaces [31,32].

The penetration of a drug into the lipid bilayer is closely related to its antioxidant activity [33,34] because it affects membrane fluidity and polarity. Indeed, numerous studies indicate that increases or decreases in membrane fluidity are responsible for the antioxidant effects of many drugs. Antioxidant drug molecules can trap free radicals or hamper their diffusion by fluidifying [1,25,35] or stiffening [36–38] the membrane, respectively. Resveratrol is able to fluidize and permeate the membrane, suggesting that it is a good antioxidant agent. Besides, the ability of resveratrol to penetrate into the membrane may depend on differently packed lipid, fluid or gel phases [1]. This behavior of resveratrol could explain the controversies regarding its location within the membrane.

In this paper, we report a systematic investigation of resveratrol incorporation into planar lipid membranes (PLMs) and formation of channel-like events using electrophysiological measurements, a new technical approach for the study of resveratrol's membrane activity, aiming to contribute to understanding the action of resveratrol on membranes, by varying PLM electrical parameters (capacitance and conductance) when the polyphenolic compound interacts with it. PLMs of different composition were used for the following reasons: they are a convenient tool to use for protein or drug incorporation, as they are a less complex membrane model system than plasma membranes; they complement studies performed with liposomes.

Incorporation into membranes and channel-like event formations are a test of resveratrol's affinity for the lipid core of PLMs, and, albeit indirectly, they can provide information on its location within the lipid double layer.
