Materials
Powdered FA and arbutin (purity >99%) were purchased from Beijing HWRK Chem Co., Ltd. Soy lecithin (phosphatidylcholine, PC; purity >98%) was purchased from Shanghai Taiwei Co., Ltd. A Strat-M® membrane was purchased from Merck Millipore (Darmstadt, Germany). Other chemical reagents were of analytical grade. Physical mixture (PM) was prepared by putting equimolar amount of ferulic acid and phospholipids into mortar and grinding the mixed material sufficiently.
Cell culture
Mouse melanoma B16F10 cells were purchased from the Cell Bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) that was supplemented with 10% fetal bovine serum (BioWhittaker, Walkersville, MD, USA) and 1% penicillin–streptomycin (Gibco BRL, NY, USA). The cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2.
Preparation of FA–PC using solvent evaporation
Screening for optimal proportion of ferulic acid and phospholipids
FA and phospholipids at molar ratios of 2:1, 1:1, 1:2, 1:3, and 1:4 were added to 100 mL round-bottom flasks and dissolved in anhydrous ethanol (FA, 2.0 mg/mL). The mixtures were stirred constantly at 40 °C for 1 h, and then the anhydrous ethanol was removed by rotary evaporation. The dried FA–PC complexes were placed in a desiccator for 24 h.
To determine the optimal ratio of FA to phospholipid, we measured the complexing rate of FA by UV spectrophotometry (UV-3150; Shimadzu, Japan). Briefly, the absorbance of prepared FA–PC samples in ethanol was determined at 323 nm. An equal amount of phospholipids dissolved in ethanol was used as a control, and a standard curve was constructed using FA. The complexing rate was expressed as mg of FA equivalents per g of dry weight.
Screening for optimal reaction temperature for FA–PC preparation
FA and phospholipids at a molar ratio of 1:1 were added to 100 mL round-bottom flasks and dissolved in anhydrous ethanol (FA, 2.0 mg/mL). The mixtures were stirred constantly at 20, 40, 60, or 80 °C for 1 h and then dried by rotary evaporation at 40 °C. Then, they were placed in desiccators in preparation for determination of FA content.
Screening for optimal reaction time for FA–PC preparation
FA and phospholipids at a molar ratio of 1:1 were added to a 100 mL round-bottom flask and dissolved in anhydrous ethanol (FA, 2.0 mg/mL). The mixtures were constantly stirred constantly at 40 °C for 15, 30 min, 1, 2, 3, or 4 h and then dried by rotary evaporation at 40 °C. Afterward, they were placed in desiccators in preparation for determination of FA content.
Screening for optimal FA concentration
FA and phospholipids at a molar ratio of 1:1 were added to 100 mL round-bottom flasks. Different volumes of anhydrous ethanol were added to each flask to yield FA concentrations of 1.0, 2.0, 4.0, 6.0, and 10 mg/mL. The mixtures were constantly stirred under 40 °C for 1 h and then dried by rotary evaporation at 40 °C. Afterward, they were placed in desiccators in preparation for determination of FA content.
Characterization of FA–PC
Differential scanning calorimetry (DSC)
DSC was carried out with a differential scanning calorimeter (Q2000; TA Instruments, USA). FA, phospholipids (PC), a physical mixture of FA and phospholipids (PM), and FA–PC, were separately loaded onto aluminum pans and heated at a rate of 10 °C/min from 25 to 300 °C under a nitrogen atmosphere for thermal analysis.
Fourier transform infrared spectroscopy (FTIR)
The infrared spectra of FA, phospholipids, PM, and FA–PC were obtained via a liquid membrane method using an FTIR spectrometer (8200, Shimadzu, Japan). The spectra were recorded at a range of 400–4000 cm−1.
Scanning electron microscopy (SEM)
The morphologies of FA, PC, PM, and FA–PC were examined under scanning electron microscope (PhenomProX; Phenom World, Netherlands) at an acceleration voltage of 10 kV. Samples were sputter-coated with gold–palladium and observed at different magnifications.
Solubility and oil–water partition coefficient
Solubility
The solubilities of powdered FA and FA–PC were determined by adding an excess of samples to 10.0 mL [10] of water or n-octanol and then shaking on a swing bed for 3 h at 37 °C. The mixtures were centrifuged at 15,000 rpm for 10 min to remove insoluble FA. Then, the supernatants were filtered through 0.45 µm membranes. Afterward, the filtrates were diluted tenfold with methanol and the FA content determined using a UV spectrophotometer (UV-3150; Shimadzu; Japan).
Oil–water partition coefficient
Samples (10 mL) of FA and FA–PC in water-saturated n-octanol were prepared and shaken. n-octanol–saturated water (10 mL) was added to each sample, and the miscible liquid was agitated for 24 h. Afterward, the samples were allowed to stand for layering. The FA concentration in each phase was determined by UV spectrophotometry (UV-3150; Shimadzu; Japan). Analyses were carried out in triplicate.
In vitro diffusion
In vitro diffusion studies were performed utilizing Franz diffusion cells (TK-20A; Shanghai Xie Kai Financial Information Service Co., Ltd.; China). In addition, we used Strat-M® membranes, which are synthetic membranes with diffusion characteristics that correlate more closely to human skin than do animal skin models [11]. The membranes were clamped between the donor and receiver chambers of the vertical diffusion cells, and the receiver chambers were filled with phosphate-buffered saline (PBS; pH 7.4) to solubilize FA or FA–PC and ensure sink conditions.
The receiver chambers were kept at 37 °C using a thermostatic water bath, and the solutions inside the receiver chambers were magnetically stirred at 500 rpm throughout the experiment. About 3.0 mg of FA or FA–PC was placed in the donor chambers. At 1, 2, 4, 8, 16, and 24 h, the solutions (0.6 mL) inside the receiver chambers were removed and filtered through 0.45 μm membrane filters. The concentration of FA in each sample was determined using a validated high-performance liquid chromatography (HPLC) method.
Chromatographic separation was carried out using an Agilent 1260LC series system (Agilent Technologies, Palo Alto, CA, USA) equipped with an online vacuum degasser, quaternary pump, autosampler, thermostatted column compartment, and diode array detection (DAD). Agilent Technologies ChemStation software for liquid chromatography (LC; B.02.01) was used, and HPLC separation was performed using an Eclipse plus-C18 column (4.6 × 250 mm, 5 μm). The detection wavelength was set to 322 nm. The mobile phase consisted of water with 0.05% acetic acid (A) and methanol (B) (40:60, v/v). The flow rate was 1.0 mL/min. The column temperature was set at 30 °C. Cumulative corrections were made to ascertain the amount of FA released at each time interval. All measurements were performed in triplicate, and the percent of cumulative FA that permeated through the membrane (%Q) was plotted as a function of time.
Inhibition of melanogenesis
B16F10 cell viability assay
Cell viability and cell proliferation were evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [2]. B16F10 cells were pretreated with 0.25, 0.5, 1.0, 2.0, 4.0 mg/mL concentrations of FA and FA–PC. After incubation for 48 h, MTT solution (final concentration: 5 mg/mL) was added, and the cells were incubated for 3 h at 37 °C. Finally, the absorbance of each sample was measured on a microplate reader at 570 nm to obtain the percentage of viable cells.
Measurement of melanin content
Melanin content was measured as described previously [6] with some modifications. B16F10 melanoma cells were seeded (2 × 105 cells/well in 3 mL of medium) in six-well culture plates and incubated overnight to allow cells to adhere. At the end of the treatment, the cells were washed with PBS and lysed with 1 M NaOH containing 10% dimethyl sulfoxide (DMSO) for 30 min at 80 °C. The absorbance (optical density; OD) was measured at 475 nm using a microplate reader. Melanin content was calculated using the following formula:
$${\text{Melanin}} \;{\text{content}} \;(\% ) = \frac{{{\text{OD}}475_{\text{sample}} }}{{{\text{OD}}475_{{{\text{blank}}\,{\text{control}}}} }} \times 100$$
Data analysis
The statistical significance of the differences between the mean measurements of each treated group and that of the control group were determined using Dunnett’s t test. P values <0.05 were considered statistically significant.