*3.2. Resveratrol Interaction with POPC PLMs*

In many different experiments on POPC PLMs, the addition of 10 or 20 µM of resveratrol to the *cis* side of the medium facing the membrane was performed at the applied voltage of 100 mV (addition voltage). After different lag times depending on the resveratrol concentration, channel-like activity appears as non-random discrete events that fluctuate between conductive or non-conductive states compatible with channel-type openings and closures with different conductance levels and frequencies. After the first channel-like event, alternating periods of channel-like activity can be observed, during which the number of events can be rigorously analyzed, followed by quiescent periods and periods of paroxystic channel-like activity that often lead to membrane destabilization until rupture. Figure 4 shows a typical example of channel-like event recordings with associated histograms of the conductance fluctuations.

In experiments at the 10 µM resveratrol concentration, channel-like activity appears after about 60 minutes' lag time at an applied voltage of 100 mV. After the first channel-like event, the applied voltage can be lowered as far as 60 mV and channel amplitude can be monitored. The channel-like activity was registered at applied voltages of ±60, ±80, ±100, and ±120 mV, each applied for 1 h, starting from 60 mV.

By doubling the resveratrol concentration to 20 µM, the lag time reduced to 20 min, after which channel-like activity appeared at an applied voltage of 100 mV, manifesting in a paroxystic manner. Paroxystic activity occurred even when the applied voltage was lowered to 80 mV; therefore, the applied voltage was lowered to 60 mV and channellike activity appeared as discrete current fluctuations with different conductance levels. Channel-like activity was monitored at applied voltages of ±40 and ±60 mV, each applied for 1 h, starting from 60 mV.

Table 1 summarizes the central conductance values (Λ<sup>c</sup> ± SE), obtained by fitting the experimental data with a Gaussian function (Figure 4), and frequency values (F ± SD) at the two different concentrations of resveratrol. Interestingly, at the 10 µM resveratrol concentration, the frequency values were higher at negative applied voltages than at positive ones, indicating a higher turnover of channel-like events.

Λ **Figure 4.** Resveratrol channel-like activity in POPC planar lipid membranes (PLMs). Representative traces illustrating channel activity of resveratrol in membranes made up of POPC with associated histograms of the conductance fluctuations. The histograms of the probability, P(Λ), for the frequency of a given conductivity unit were fitted by a Gaussian which is shown as a solid curve. Experiments were performed in the presence of 10 µM (top trace) and 20 µM (bottom trace) of resveratrol added to the *cis* side, while the aqueous phase contained 0.1 M KCl (pH 7) and T = 23 ± 1 ◦C. Applied voltage was set to 60 mV (top trace) and 40 mV (bottom trace).

**Table 1.** Characteristic parameters of resveratrol channel-like event in POPC PLM. The mean conductance (Λ<sup>c</sup> ± SE) and frequency (F ± SD) of resveratrol channel-like events at different applied voltages. The minimum and maximum number of channel-like events considered (N) out of a total number of channel-like events considered (Nt) was: 130 < N < 375, Nt = 1640, at the resveratrol concentration of 10 µM. The minimum and maximum number of channel-like events considered (N) out of a total number of channel-like events considered (Nt) was: 280 < N < 622, Nt = 1757, at the resveratrol concentration of 20 µM.


Another parameter that we calculated is the duration of observed channel-like events. The duration of channel-like events was within a range from 1.25 to 2.25 s and from 0.75 to 1.25 s when the resveratrol concentration was 10 and 20 µM, respectively. The lifetime for two resveratrol concentrations, although not significantly different, indicates that channellike events were more stable at lower resveratrol concentrations than at higher ones.

The results obtained with POPC PLMs indicate that resveratrol is able to penetrate into the hydrophobic core of PLMs and to form channel-like events. The lag time, the applied voltages at which the channel-like activity manifests, and the shorter lifetimes decrease on increasing resveratrol concentration, in agreement with its poor water solubility and its partition coefficient and with the concept that an appropriate resveratrol/lipid ratio, is required to permeabilize the membrane.

Our experimental apparatus can measure conductance and capacitance simultaneously. Capacitance is related to the property of a membrane to act as a capacitor and is inversely proportional to its thickness. The capacitance parameter is considered to be the best tool for probing the stability and formal goodness of the double lipid layer before and after any experiment concerning the incorporation of a different substance can be conducted [47].

In all experiments, before the addition of resveratrol, the basic capacitance of the POPC PLMs was in the range of 0.27–0.28 µF/cm<sup>2</sup> , remaining constant. After the addition of resveratrol, we observed that the capacitance decreased during the lag time, then increased when the first channel-like events appeared, reaching higher values than those of basic capacitance; these values remain almost constant all the way through to the end of the experiment. This behavior was independent of the resveratrol concentrations used in this study.

Table 2 reports the mean capacitance (C ± SE) values calculated at different times before and after the addition of resveratrol. To standardize the results, capacitance was measured just before the addition of resveratrol (T0), 70% of the way through the lag time (T1), at the end of the lag time when the channel-like activity appears (T2), and after an average of 1 h from the first channel-like event when the capacitance signal was almost constant (T3). The capacitance variation observed could be due, initially, to resveratrol adsorption to the planar lipid bilayer and, subsequently, to its insertion into the bilayer once an appropriate resveratrol/lipid ratio had been reached.

**Table 2.** Capacitance variation in POPC PLM. Mean values of the membrane capacitance (C ± SE) calculated at T0 (just before the addition of resveratrol), T1 (at the 70% of lag time), T2 (at the end of lag time), and T3 (after an average of 1 h from first channel-like event appearance). The mean value was obtained from at least three experiments.

