4.4. Evaluation of Free Radical Scavenging Activity

The studies on the antioxidant activity of betulin and its derivatives were carried out using the DPPH [13,47,48], ABTS [13,47] and the Folin–Ciocalteu [13,47,49] methods. The absorbance at λ 517 nm (in the case of the DPPH method), 734 nm (in the case of the ABTS method), and 765 nm (in the case of the Folin–Ciocalteu method) working solutions was adjusted to 1.00 ± 0.02. Measurements were taken using a Spectroquant, model Pharo 300 (Merck, Germany), in triplicate for each tested sample. The efficiency of neutralization of DPPH radicals by betulin and its derivatives was expressed in the form of TEAC (Trolox Equivalent Antioxidant Capacity) factor, determining the concentration of Trolox with identical antioxidant capacity. TEAC values were calculated from absorbance measurements using the calibration curve method. The results were expressed as TEAC in millimoles of trolox per volume of sample (mmol TE/dm³). In the case of the Folin–Ciocalteu method, the antioxidant activity was expressed in mmol GA/dm³.

In the next stage of the compound characterized by the highest degree of free radical scavenging DPPH (compound 8), penetration through the pig skin and its accumulation in the skin were evaluated. For comparison, penetration studies of betulin were also carried out.

The antioxidant activity of the tested compounds was evaluated using a modified DPPH method. The procedure was as follows: to 2.85 mL of the DPPH radical solution (at concentrations of 0.3 mmol/dm³) dissolved in 96% (v/v) ethanol to 0.15 mL of the tested compounds (at the concentrations of 0.226 mmol/dm³) was added, and then the tube was incubated for 10 min at room temperature, followed by spectrophotometric measurements at λ = 517 nm.

The antioxidant activity of the tested samples was calculated according to the following formula:

where: %RSA—antioxidant activity, A₀—mean value of the absorbance of the ethanol solution of DPPH containing 0.15 mL of the ethanol, A_p—mean value of absorbance of the ethanol solution of the DPPH radical containing 0.15 mL of the tested compound.

The antioxidant activity of the tested compounds was evaluated by the ABTS method, using a solution of ABTS (at concentrations of 7 mmol/dm³) in an aqueous solution of potassium persulfate (at concentrations of 2.45 mmol/dm³) as a stock solution. The solution was incubated for 24 h, at the temperature of 4 °C and then diluted with 50% (v/v) methanol. The antioxidant activity was measured as follows: to 2.5 mL of a stock solution of ABTS to 0.25 mL of the tested compound (at the concentrations of 0.226 mmol/dm³) was added, and then the tube was incubated for 6 min at room temperature. Spectrophotometric measurements were carried out at λ = 734 nm.

The antioxidant activity was calculated from the equation:

where: %RSA—antioxidant activity, A₀—absorbance of the stock solution ABTS containing 0.25 mL of the ethanol, A_p—absorbance of the stock solution ABTS containing 0.25 mL of the tested compound.

The total content of phenolic compounds found in the tested samples was evaluated using the Folin–Ciocalteu method. Two milliliters of Folin–Ciocalteu reagent in 1.8 mL of water was dissolved in a dark bottle, then the solution was incubated for 60 min (at room temperature). The antioxidant activity was measured as follows: 1.35 mL of distilled water and 1.35 mL of sodium carbonate solution (at concentrations of 0.01 mol/dm³) were introduced into the tube, with 0.15 mL of the prepared Folin–Ciocalteu solution and 0.15 mL of an ethanol solution containing the tested compound (at a concentration of 0.226 mmol/dm³), and then the tube was incubated for 15 min at room temperature, followed by spectrophotometric measurements at λ = 765 nm.