## 2 Materials and Methods
### The Skin Permeability of Hydrogels and Organogels and the Cumulative Mass of Active Compounds  
The skin permeability for formulations (hydrogels and organogels containing silymarin, bio-ferment, and extract) of H-S, H-B, H-E, O-S, O-B, and O-E was assessed in Franz diffusion cells (Phoenix DB-6, ABL&E-JASCO, Wien, Austria) that consisted of a 1 mL donor chamber and a 10 mL acceptor chamber. The acceptor chamber, equipped with a magnetic stirring bar, was filled with PBS solution at a pH of 7.4. The acceptor chamber was maintained at a constant temperature of 37 \(\pm\) 0.5 \({}^{\circ}\)C. Prior to commencing the examination, Franz diffusion cells were allowed to equilibrate at 37 \({}^{\circ}\)C for 15 min. The study utilized porcine skin owing to its comparable permeability qualities to human skin. The skin originated from a nearby abattoir. A fresh portion of skin from the abdomen was washed several times with a solution of PBS. Skin with a thickness of 0.5 mm was cut with a dermatome, and then it was enveloped in aluminum foil and frozen at a temperature of \(-\)20 \({}^{\circ}\)C for a maximum of 3 months [32; 39; 40]. Before the study, the skin was thawed at room temperature for about 30 min, and then it was soaked in PBS solution for 15 min to hydrate it. Subsequently, the skin was affixed to Franz diffusion cells. The integrity of the skin was checked 1 h after its installation in the Franz diffusion chamber (SES GmbH Analyse System, Bechenheim, Germany). In order to achieve this objective, the measurement of skin impedance was conducted using a 4080 m LCR (Conrad Electronic, Bechenheim, Germany) operating in parallel mode at 120 Hz (k\(\Omega\) error \(<\) 0.5%). To take the measurement, the tips of the probes were immersed in the donor and acceptor chambers filled with the PBS solution. The membranes have an electrical resistance that is more than 3 k\(\Omega\), which is equivalent to the resistance observed in normal human skin [34].  
Formulations (H-S, H-B, H-E, O-S, O-B, and O-E) were positioned within the donor chamber. All donor chambers were closed with a plastic stopper to prevent excessive evaporation of the vehicle. The aforementioned tests were conducted for a duration of 24 h. A 0.5 mL portion of the solution in the acceptor chamber was taken at specified intervals (0.5, 1, 2, 3, 4, 5, 8, and 24 h), and then augmented with a new aliquot of buffer at an identical pH [35]. The samples underwent analysis using high-performance liquid chromatography (HPLC). Upon the conclusion of the permeation experiment, the skin was extracted to estimate the amount of silymarin accumulated in the skin.  
It was assessed whether the active substance contained in formulations penetrated the skin. Active substance contained in H-S, H-B, H-E, O-S, O-B, and O-E formulations penetrating the acceptor chamber was determined via HPLC [34]. The experiment was conducted with a Franz diffusion chamber, in which the donor phase consisted of the formulations tested. The acceptor phase consisted of a PBS solution, because it corresponds to systemic conditions, exhibits isotonic properties, and resembles conditions prevailing in the deeper layers of the skin [40]. After 24 h of the experiment, it was observed that the active substance contained in formulation penetrated the skin.