The following reagents were used in the work: sodium chloride (NaCl), HEPES, NaOH, ethanol, dimethyl sulfoxide (DMSO), Triton X-100, Sephadex G-50, calcein, sorbitol, calcium chloride (CaCl2), polyethylene glycol with a molecular weight of 8000 Da (PEG-8000), quercetin, myricetin, rutin, betulin, lupeol, 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), cholesterol (CHOL), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine) from Sigma (USA).

Grapefruit seed extract (EGS), sea buckthorn leaf extract (ESBL) and chaga extract (EC) were provided by Evalar CJSC. Testing was carried out for three samples of each extract, representing independent extraction series.

Investigation of the ability of the tested extracts and their components to inhibit the fusion of lipid vesicles mediated by various inducers. Monolamellar lipid vesicles from mixtures of DOPC/CHOL (80/20 mol %) and DOPC/DOPG/CHOL (40/40/20 mol %) loaded with a fluorescent marker calcein were formed using a mini extruder from Avanti Polar Lipids (USA). The solutions of lipids in chloroform were placed in a vial and the solvent was removed by a nitrogen stream. The resulting lipid film was hydrated with a buffer (35 mM calcein, 10 mM HEPES-NaOH, pH 7.4) and after five times freezing–thawing the mixture was passed through a polycarbonate membrane (Nucleopore TM, USA) with a pore diameter of 100 nm for 13 times to obtain a homogeneous population of large single-layer liposomes. The calcein that was not entrapped in the vesicles was removed by gel filtration on a column with Sephadex G-50. A calcein-free buffer (150 mM NaCl, 10 mM HEPES-NaOH, pH 7.4) was used as an eluent. Fluorescence of calcein inside liposomes at a concentration of 35 mM was self-quenched. Calcium (40 mM CaCl2) and polyethylene glycol with a molecular weight of 8000 Da (PEG-8000 at a concentration of 20 wt %) were used to induce the fusion of DOPC/DOPG/CHOL and DOPC/CHOL liposomes, respectively [19–22].

The samples of extracts from the initial solutions in water or DMSO were added into a suspension of liposomes up to a concentration of 100 µg/mL.

The fluorescence intensity of calcein outflowing from the liposomes into the solution medium during their fusion was measured with a Fluorat Panorama-02 spectrofluorimeter (at an excitation wavelength of 490 nm and emission of 520 nm). At the end of each experiment, Triton X-100 was introduced into the solution. At a concentration of 10 mM, this detergent destroyed all liposomes in the suspension, releasing the marker trapped by vesicles into the medium.

The index of inhibition of lipid vesicle fusion (II) by the tested extracts was calculated as :

$$II = \frac_}}}} - R_}}}}}}_}}}}}}\,\, \times \,\,100\% ,$$

(1)

where RFInd and RFInh represented the average maximum leakage of the fluorescent marker from vesicles induced by the introduction of calcium chloride or PEG-8000 in the absence and in the presence of the tested extracts, respectively.

The values of RF (%) (RFInd and RFInh) were determined by the formula:

$$RF = \frac _}}} - _}}}}^} - _}}}\,\, \times \,\,100\% ,$$

(2)

where Ii and I0 were the fluorescence intensities of the solution in the presence and absence of the tested extracts, Imax was the fluorescence intensity of the solution after the addition of Triton X-100 (multiplier 1.1 was introduced to account for dilution of the sample with an aqueous detergent solution). RFInh was evaluated considering a possible intrinsic effect of extracts on the permeability of liposomes for the marker (calcein leakage due to the disordering of lipids under the action of extracts).

The kinetics of marker release under the action of fusion inducers before and after the introduction of the tested extracts was characterized by the time of e-fold increase in relative fluorescence.

For each type of extract, nine independent repetitions were performed with samples from three different batches. The parameters characterizing the effect of individual components of extracts on membrane fusion were determined by calculating the arithmetic mean values obtained in 2–3 independent experiments.

To prove the statistical significance of the detected differences in the average RF values before and after the addition of extracts or their components, the nonparametric Mann–Whitney–Wilcoxon criterion (*p ≤ 0.01) was used.

Confocal fluorescence microscopy of giant liposomes. Visualization of changes in the behavior of vesicles before and after the introduction of PEG-8000 and chaga extract into the suspension was performed using confocal fluorescence microscopy. Giant unilamellar vesicles were prepared from the mixture of DOPC/CHOL (80/20 mol %) and 1 mol % fluorescent lipid probe 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(rhodamine lizzamine) by electroformation (standard protocol, 3 V, 10 Hz, 1 h, 25°C) using a commercially available Nanion vesicle prep pro device (Germany). The resulting suspension of liposomes contained 0.5 mM of lipid in 1 M sorbitol solution. To induce vesicle fusion, PEG-8000 was added into the suspension to a concentration of 10 wt % and incubated for 5–10 min at room temperature (25 ± 1°C). EC (35 µg/mL) and PEG-8000 (10 wt %) were sequentially added into the experimental samples with incubation at each stage for 10 min. Liposomes were observed through a 100×/1.4 HCX PL immersion lens in a Leica TCS SP5 Apo confocal laser system (Leica Microsystems, Germany). The observations were carried out at 25°C. Fluorescence of 1,2‑dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Rhodamine lizzamine) was excited at 543 nm (Helium–neon laser).

Differential scanning microcalorimetry of lipid vesicles modified with extract components. Giant unilamellar liposomes were formed from DPPG by electroformation using the Nanion vesicle prep pro device (Germany). An alternating voltage with an amplitude of 3 V and a frequency of 10 Hz was applied to the glasses for 1 h at 55°C. The lipid concentration was 3 mM. Quercetin, myricetin, and rutin were added into the experimental samples until the lipid : flavonol ratio reached 10 : 1. Thermograms of liposomal suspensions were obtained using a µDSC7 differential scanning microcalorimeter (Setaram, France). Reproducibility of the temperature dependence of the heat capacity was achieved by reheating the sample immediately after cooling at a constant rate of 0.2°C/min. The peaks on the thermograms were characterized by the maximum temperature of the main phase transition of DPPG (Tm) and the half-width of the main peak (T1/2). The changes in these parameters made it possible to evaluate the effect of flavonols on the packing density of membrane lipids.

The parameters characterizing the effect of flavonols on the thermotropic behavior of lipids were determined by calculating the arithmetic mean of the values obtained for 2 independent series of liposome preparation.

The values of RF, t, II, ΔTm, and ΔT1/2 were presented as mean ± standard error of the mean (mean ± SE).