Trichloroacetic acid tca

The anti-protease activity was determined as follows [30 (link)]: 10 μL of skin-mucus sample (undiluted) or 5 μL of sample serum (1:2 dilution) were mixed with a standard trypsin solution (20 μL; 5 mg mL⁻¹, Lonza, Basel, Switzerland) and incubated for 10 min (RT). Azocasein solution (60 μL; 1% (w/v), Sigma-Aldrich, St. Louis, MO, USA) was added to the mixture and incubation continued for 1 h (RT). Following this, trichloroacetic acid (TCA) (100 μL, 10% (w/v), Applichem, Darmstadt, Germany) was added and incubated for 30 min. The mixtures were centrifuged (1900× g, 10 min), and 100 μL of each supernatant were transferred to a clean flat-bottom 96-well transparent microplate containing 100 μL of 1 M NaOH per well. The absorbance was measured at 450 nm. Each sample was expressed as the percentage of trypsin inhibition. The percentage inhibition of trypsin activity was calculated by comparing it with a 100% control sample, in which buffer replaced the serum, while for the negative control, buffer replaced both serum and trypsin, based on the equation % Trypsin inhibition = (OD_Trypsin − OD_Sample)/OD_Trypsin × 100.

The cytotoxicity profile of propolis extracts was assessed by the SRB assay. Briefly, 5 × 10³ HaCaT cells per well were seeded in 96-well microplates, cultured for 24 h and then treated with different concentrations of propolis extracts (0–200 μg/mL) for 24 h. Then, the cells were fixed by adding 50% (w/v) cold trichloroacetic acid (TCA) (Applichem, Darmstadt, Germany) in each well and then were stained with 0.4% (w/v) sulforhodamine B (SRB) (Sigma-Aldrich, Dorset, U.K.) in 1% (v/v) acetic acid (Scharlau, Barcelona, Spain). The bound dye was dissolved in 10 mM Tris base (Sigma-Aldrich, Dorset, U.K.) and the absorbance was measured at 570 nm using a multi-plate reader (Tecan, Männedorf, Switzerland). The % cell survival was calculated using the formula:
The EC₅₀ values (effective concentration that causes 50% decrease in cell viability) of all propolis extracts were determined by regression analysis using a four-parameter logistic curve with the Sigma Plot Software v.10 (Systat, San Jose, CA, USA).

The viability of cancer cells after treatment with the juice was determined using the Sulforhodamine B (SRB) assay, as previously described with few modifications [14 (link)]. SRB is a dye that binds to basic amino acids of cellular proteins and, then, the number of viable cells is estimated with colorimetric evaluation [15 (link)]. Cells were plated in 96-well plates and treated with different concentrations of the juice (0.0007–1% v/v). Then, the cells were fixed with the addition of 25 μL of 50% (w/v) cold trichloroacetic acid (TCA) (Applichem, Darmstadt, Germany) to the growth medium and incubation of the plates at 4 °C for 1 h. The cells were washed five times with tap water and then stained with 50 μL of 0.4% (w/v) SRB dye (Sigma-Aldrich, St Louis, MO, USA) in 1% (v/v) acetic acid (Scharlau, Barcelona, Spain) for 30 min at room temperature. Next, the cells were rinsed five times with 1% (v/v) acetic acid to remove the unbound dye. The fixed, stained plates were allowed to air-dry and afterwards the bound dye was solubilized by adding 100 μL of 10 mM Trizma base (Sigma-Aldrich, St Louis, MO, USA) for at least 5 minutes. Absorbance was measured at 570 nm using an ELISA plate reader (Sunrise, Tecan, Männedorf, Switzerland) and the percent cellular survival was calculated using the formula: