## 2 Materials and Methods
### Determination of Tannin Content in Herbal Infusions and Ethanol Extracts  
The tannin analysis was conducted using a method described in the European Pharmacopoeia, 8 th edition [39]. Freeze-dried samples were prepared using 0.100 g of dry herbal material, and in the case of liquid ethanol extracts, 2 mL was treated according to the procedure described in Eur. Pharm 8 [39]. In brief, a 2 mL filtrated solution was mixed with 1 mL of phosphomolybdotungstic reagent (10% in ethanol) and 10.0 mL of water, then diluted to 25 mL with a sodium carbonate solution (29 g/100 mL). After a 30 min incubation in the dark, the absorbance was measured at 760 nm using a UV/VIS spectrophotometer (Mettler-Toledo UV7, LabX(tm), Greifensee, Switzerland). Pyrogallol (50 mg/100 mL) served as the standard. The results were calculated as percentages of tannins expressed as pyrogallol in dried aerial parts. All measurements were made in triplicate. The following formula was used to calculate the percentage of tannin content:  
\[\text{Tannin content per g of dried plant, }\%=\frac{62.5\times(\text{A}_{1}-\text{A}_{2})\times \text{m}_{2}}{\text{A}_{3}\times\text{m}_{1}},\]  
where A\({}_{1}\) is the absorbance of the total polyphenols; A\({}_{2}\) is the absorbance of polyphenols not adsorbed by the hide powder; A\({}_{3}\) is the absorbance of standard pyrogallol; m\({}_{1}\) is the mass of the sample to be examined, in grammes; and m\({}_{2}\) is the mass of pyrogallol, in g.  
Determination of Antioxidant Activity Using a DPPH (2,2-Diphenyl-1-picrylhydrazyl) Radical Scavenging Assay  
DPPH was used to evaluate the free radical scavenging (antioxidant) potential of the extracts. DPPH radical scavenging activity was assessed according to the methodology introduced by [41], with certain adjustments. A DPPH solution was prepared by dissolving 0.0118 g of 2,2-diphenyl-1-picrylhydrazyl in 300 mL of methanol, and it was stored in a dark place. For the assay, varying volumes (1 to 20 \(\mu\)L) of the ethanol extract or 16 mg of freeze-dried extract dissolved in 1 mL of water and then diluted twofold four times, diluted in 10 \(\mu\)L to 29 \(\mu\)L of methanol, were combined with 3 mL of the DPPH solution. The samples were incubated in darkness at room temperature for 15 min, and the reduction in absorbance at 515 nm was measured using a UV/VIS spectrophotometer (Mettler-Toledo UV7, LabXtm, Greifensee, Switzerland). A Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) solution (20 mg/10 mL in methanol) served as the standard, while methanol was used as a control. The samples were tested at various concentrations to determine the IC50 (the concentration that reduces the absorbance of DPPH by 50%). The radical scavenging activity was calculated using the following formula:  
\[\text{S}\text{c}\text{c}\text{v}\text  
and the column temperature was maintained at 30 \({}^{\circ}\)C. The injection volume was 4 \(\mu\)L, and the gradient programme was set as in Table 1.  
Mass spectrometric analysis was performed using an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a heated electrospray ionisation (HESI-II) probe (Thermo Fisher Scientific). The instrument operated in both negative and positive ion modes within the \(m/z\) range of 100 to 1500. For the mass spectrometer, the spray voltage was 2.5 kV (\(-\)); the sheath gas flow rate was 50; the auxiliary gas flow rate was 10; the capillary temperature was 325 \({}^{\circ}\)C; the probe heater temperature was 350 \({}^{\circ}\)C; the S-lens RF level was 70; and the scan mode was full MS (resolution 60,000) and ddMS2 (15,000), with standard collision energy and HCD normalised collision energy set to 30%. Data were processed using the software Xcalibur 4.6 (Thermo Fisher Scientific, Waltham, MA, USA) for instrument control and data handling. Compound profiling was applied to the UHPLC- HRMS raw files of the studied extracts using the TraceFinder 5.1 software (Thermo Fisher Scientific, Waltham, MA, USA).