## 2 Materials and Methods
### Preparation of Mulberry Extract  
Fresh mulberry fruits (5 g) were extracted with 5 mL acidified methanol (trifluoroacetic acid (TFA), 0.1%) by homogenization (Heidolph homogenizer, Silent Crusher M) at 12,000 rpm for one minute. The homogenized samples were then centrifuged (NUVE NF 200 BENCH TOP CENTRIFUGE, Turkey) at 5000 rpm for 20 min. The pellet was subjected to repeated extraction until complete decolorization of the solvent. The collected supernatant was concentrated, by evaporation of methanol, in the vacuum rotary evaporator (Heidolph Rotary Evaporator, Laborota 4000 rotavapor, Schwabach, Germany) at 40 \({}^{\circ}\)C, according to [PERSON] et al. (2011) [27] with minor modifications [6]. The _Morus nigra_ extract (MnE) was freeze-drying using Christ Alpha 1-2 LDplus freeze dryer (Osterode am Harz, Germany) and used for the cell culture analysis. HPLC-DAD-MS (ESI\({}^{*}\)) Characterization of the Extract from the Point of View of Anthocyanin Content  
For the separation and identification of phenols by HPLC, 0.5 g of mulberries were extracted with acidified methanol with 1% HCl of 37% by vortexing for 1 min with Heidolph Reax top vortex (Heidolph Instruments GmbH & Co., Schwabach, Germany), sonicated  
Figure 1: Experimental design. MnE—_M. nigra_ L. extract; MnL—liposomes loaded with _M. nigra_ L. extract (25 ug/mL), L—empty liposome. for 15 min in the Elmasonic E 15 H sonication bath, and centrifuged at 10,000 rpm, for 10 min and 40 \({}^{\circ}\)C in the Eppendorf AG 5804 centrifuge, SUA. The supernatant was filtered through filter paper and kept in the dark. The pellet was subjected to repeated extraction, under the conditions specified above, until complete discoloration. The cumulative extract was concentrated, by evaporation at low pressure on the Heidolph rotary evaporator, to a volume of 2 mL, it was filtered through a 0.45 \(\upmu\)m Chromafil Xtra nylon filter and 20 \(\upmu\)L were injected into the HPLC system [27]. Identification of the compounds in the black mulberry fruit extract was performed using an HP-1200 liquid chromatograph equipped with a quaternary pump, autosampler, DAD detector, and single-quadrupole API electrospray MS-6110 detector (Agilent Technologies, Santa Clara, CA, USA). For the detection of phenolic compounds, full scan ESI positive ionization mode was used in the following working conditions: capillary voltage 3000 V, temperature 3500 C, nitrogen flow 7 L/min and \(m/z\) 120-1200. The column was a Kinerex XB-C18 (5 \(\upmu\)m; 4.6 \(\times\) 150 mm i.d.) from Phenomenex, Torrance, CA, USA. The mobile phase A was water acidified by 0.1% acetic acid and mobile phase B was acetonitrile acidified with 0.1% acetic acid. The following multistep linear gradient was applied: 0 min, 5% B; 0-2 min, 5% B; 2-18 min, 5-40% B; 18-20 min, 40-90% B; 20-24 min, 90% B; 24-25 min, 90-5% B; 25-30 min, 5% B. Total analysis time was 30 min, flow rate 0.5 mL/min and oven temperature 25 \(\pm\) 0.5 \({}^{\circ}\)C.  
Mass spectrometric detection of positively charged ions was performed using scanning mode. The applied experimental conditions were: gas temperature 350 \({}^{\circ}\)C, nitrogen flow 7 L/min, capillary voltage 3000 V and \(m/z\) 120-1200. Chromatograms were recorded at wavelength \(\lambda\) = 280, 340 and 520 nm, and data acquisition and interpretation of results was done using Agilent ChemStation software (B.02.01 SR2, Santa Clara, CA, USA). Phenolic compounds were identified by comparing retention time, UV-Vis absorption and mass spectra with those of standard compounds and literature data [1,28]. Based on the spectral characteristics of phenolic compounds, the wavelength \(\lambda\) = 280 nm is specific for some phenolic acids, flavanol monomers and polymers, while \(\lambda\) = 340 nm for hydroxcinaminatic acids and flavonols, and \(\lambda\) = 520 nm for anthocyanins.