*3.3. Resveratrol Interaction with POPC:Ch PLM*

The addition of cholesterol to the POPC solution used to form membranes results in channel-like activity, indicating that the sterol does not prevent the insertion of resveratrol molecules into PLMs made up of POPC:Ch.

The addition voltage of resveratrol was 100 mV both for experiments at a concentration of 10 µM and for those at a concentration of 20 µM. The lag time and applied voltage in which the channel-like activity appears depend on the resveratrol concentration used in the two series of experiments.

After the addition of resveratrol to the *cis* side of the medium facing the membrane, channel-like activity manifests, first of all, as a paroxystic activity then characterized by current jumps compatible with channel-type openings and closures with different conductance levels followed, sometimes, by quiescent periods. Figure 5 shows an example of chart recordings of resveratrol's channel-like activity when incorporated into

POPC:Ch PLMs at the applied voltage of 80 and 40 mV with associated histograms of the conductance fluctuations.

Λ **Figure 5.** Resveratrol channel-like activity in POPC:Ch PLMs. Representative traces illustrating channel activity of resveratrol in membranes made up of POPC:Ch (65:35, w:w) 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 80 mV (top trace) and 40 mV (bottom trace).

> In the set of experiments at 10 µM of resveratrol, channel-like activity appears after a lag time of 6–7 h at an applied voltage of 120 mV (activation voltage) as a paroxystic activity, after which the applied voltage was lowered down as far as 80 mV where the channel-like event amplitude could be monitored. The channel-like activity was recorded at applied voltages of ±80, ±100, and ±120 mV, each applied for 1 h, starting from 80 mV.

> In the set of experiments at 20 µM of resveratrol, channel-like activity manifests as a paroxystic activity after a lag time of 2 h at an applied voltage of 100 mV followed by membrane rupture. After PLM breakage and withdrawal, the channel-like activity occurred spontaneously after a lag time of about 15 min at an applied voltage of 40 mV. The channel-like activity was monitored at applied voltages of ±40 mV, each applied for 90 min, starting from 40 mV. The application of voltages higher than ±40 mV (±60 and ±80 mV) determined paroxystic activity followed by destabilization of the membrane.

> Table 3 reports the biophysical and statistic parameters (Λ<sup>c</sup> ± SE and F ± SD, respectively) of the channel-like events obtained under the two different experimental conditions. It is important to note that, at a resveratrol concentration of 10 µM, the central conductance and frequency values seem to be independent of applied voltages both at positive and at negative voltages.

> The comparatively higher lag times and applied voltages observed in these experimental sets than those for POPC PLMs may be due to the effect of cholesterol on acyl carbon chains that are more closely packed. Resveratrol penetrates the hydrophobic core of the membrane less easily.

> As was done for POPC PLMs, we calculated the lifetime of channel-like events formed by resveratrol. The duration of channel-like events was within a range from 1.25 to 1.75 s when the resveratrol concentration was 10 µM, while, at 20 µM, the duration of channel-like events was 1.75 s at applied voltages of ±40 mV.

**Table 3.** Characteristic parameters of resveratrol channel-like event in POPC:Ch 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: 186 < N < 516, 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: 265 < N < 337, Nt = 602, at the resveratrol concentration of 20 µM.


The capacitance behavior recorded in the experiments with POPC:Ch PLMs was similar to that observed for POPC PLMs at the two different resveratrol concentrations. Table 4 reports the mean capacitance values at four different times, T0, T1, T2, and T3. It is interesting to note that the low values of capacitance measured at 70% of lag time (T1) could be due to resveratrol adsorption to the POPC:Ch PLM before its insertion into the bilayer, similarly to that observed for POPC PLMs.

**Table 4.** Capacitance variation in POPC:Ch PLM. Mean values of the membrane capacitance (C ± SE) calculated at T0, T1, T2, and T3, for which meanings are reported in the legend to Table 3. The mean value was obtained from at least four experiments.

