## 2 Materials and Methods
### Study Group and Skin Biophysical Parameter Evaluation  
The effect of the cream containing _K. elatine_ cell extract on skin biophysical parameters was assessed in a randomised and placebo-controlled study involving 40 participants. Eligible participants were healthy adult females aged 40 to 49 years. Exclusion criteria included pregnancy or breastfeeding, the presence of skin diseases, and the use of systemic medications with strong pharmacological activity. The block randomisation method was applied to ensure the size of each group (placebo and treatment) was similar throughout the entire study.  
Skin biophysical parameters assessed in this study included skin hydration, transeip-dermal water loss (TEWL), erythema intensity, melanin intensity, skin surface pH, and skin structure, following a previously described protocol [25]. Non-invasive skin approaches were used to measure the parameters using the Courage & Khazaka MPA-9 (Courage+Khazaka electronic GmbH, Cologne, Germany) and Nati Skin Analyzer (Beauty of Sciences, Wroclaw, Poland). The Corneometer(r) CM 825 probe (Courage+Khazaka electronic GmbH, Cologne, Germany) was used to measure the electrical capacitance of the _stratum corneum_ (skin hydration) by operating at 40-75 Hz. TEWL was measured using the Tewameter(r) TM 300 (Courage+Khazaka electronic GmbH, Cologne, Germany). Erythema index and melanin index were evaluated using the Mexameter(r) MX 18 probe (Courage+Khazaka electronic GmbH, Cologne, Germany). The Skin-pH-meter PH 908 probe (Courage+Khazaka electronic GmbH, Cologne, Germany) was used to assess the acidity of the skin surface. Lastly, the sebum level and luminosity of the skin surface were determined using Sebumeter(r) SM 815 and Skin-Glossymeter GL 200 (Courage+Khazaka electronic GmbH, Cologne, Germany), respectively. The Nati Skin Analyzer evaluates skin parameters physically and optically using UV and white light. Skin structure, wrinkle depth, vascular lesions, and discolouration were analysed using the Nati Skin Analyzer. The device grades the parameters using red, yellow, and green spectra (Figure 2B). The colours correspond to numerical scores based on specific skin parameter evaluations (Table 1).  
Figure 1: Schematic process from in vitro culture to product formulation containing _K. elatine_ cell extract.  
Figure 2: Designated area for measurement (**A**) and a graphical interpretation of the results (**B**) obtained after skin analysis with the Nati Skin Analyzer.  
The tests were performed in a controlled environment at 22 \(\pm\) 3 \({}^{\circ}\)C and a relative humidity of 45 \(\pm\) 2%. The measurements were carried out on a designated area (left cheek) to ensure repeatability (Figure 2A). The probes were placed on the skin surface, and each parameter was assessed in three technical replications at baseline and after six weeks of application. The results were calculated and presented as mean \(\pm\) standard deviation (SD).  
Furthermore, the sensory evaluation of the cream containing _K. elatine_ cell extract was assessed through surveys that the participants completed during the final measurement (six weeks after application). The evaluated attributes included the following: colour, consistency, fragrance notes, fragrance intensity, cream texture, spreadability, absorption, effectiveness, and overall characteristics. A 5-point scale was used to evaluate colour and fragrance notes, ranging from (1) very unpleasant, (2) unpleasant, (3) neutral, (4) pleasant, to (5) very pleasant. A similar range scale was applied to spreadability, absorption, efficiency, and overall characteristics of the creams (1--very bad, 2--bad, 3--medium, 4--good, 5--excellent). Moreover, fragrance intensity and cream consistency were evaluated using a 3-point scale: (1) not intense enough, (2) appropriate, and (3) too intense for fragment intensity; and (1) too runny, (2) appropriate, and (3) too thick for cream consistency.