# Antioxidant Capacity of Honey Enriched by Wildflowers
## 2 Materials and Methods
### Honey Samples and Experimental Conditions  
Two varieties of honey, namely multiforal and rapeseed (_Brassica napus_ L.), were used for the analysis. These honey samples were sourced from apiaries situated in a mountainous agro-forestry region within the Carpathian Foothills in southeastern Poland, located at precisely 49\({}^{\circ}\)48\({}^{\prime}\)09\({}^{\prime\prime}\) N 21\({}^{\circ}\)31\({}^{\prime}\)53\({}^{\prime\prime}\) E. The floral origin of the samples was specified by the beekeepers based on the hive's location and available floral sources. The honey samples were divided into sterile glass containers equipped with lids. Plant material, either in the form of ground-dried plants or ethanol extracts, was then introduced into the containers. The mixture was carefully and thoroughly stirred to ensure proper integration. The plant addition was 1%, 2%, or 4% (\(w/w\)). For the extracts, a volume corresponding to an identical mass of plant material was used. The control samples were honey of both varieties without any plant material added. The honey samples were kept in a dark place  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & & \multicolumn{3}{c}{**Samples Enriched**} & \multicolumn{3}{c}{**Samples Enriched**} \\  & & **by Dried Flowers** & & & **by Extract** & \\ \cline{2-7}  & **1\%** & **2\%** & **4\%** & **1\%** & **2\%** & **4\%** \\ \hline
**Honey (g)** & 99.0 & 98.0 & 96.0 & 98.0 & 98.0 & 98.0 \\ \hline
**Dried flowers (g)** & 1.0 & 2.0 & 4.0 & - & - & - \\ \hline
**Extract (mL)** & - & - & - & 0.5 & 1.0 & 2.0 \\ \hline
**Ethanol 50\% (mL)** & - & - & - & 1.5 & 1.0 & 0.0 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Ingredients used to prepare the honey samples.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Latin name** & _Pulmonaria_ & _Malva_ & _Primula_ & _Tussilogo_ & _Bellis_ & _Sambucus nigra_ L. \\  & _officialnis_ L. & _sylvestris_ L. & _veris_ L. & _farfar_ L. & _perntnis_ L. & _Sambucus nigra_ L. \\ \hline
**Common name** & Lungwort & High mallow & Cowslip & & & \\  & & primrose & & & & \\ \hline
**Collection time** & April & June & April & May & May & June \\ \hline \hline \end{tabular}
\end{table}
Table 1: Plant species used for honey enrichment.  
at room temperature (21 \(\pm\) 1 \({}^{\circ}\)C) until analysis. The sample storage time was 3 months, and the samples were stirred once every 30 days. Before further preparation procedures, the honeys were liquefied using an ultrasonic bath (Sonic-6D, Polsonic, Warsaw, Poland) at 40 \({}^{\circ}\)C for 1 h. For testing, 5 g portions from the honey samples were moved into sterile plastic flasks with lids and dissolved in 20 mL of water with a 1% acetic acid addition. The aqueous solutions were again placed in an ultrasonic bath for 30 min (40 \({}^{\circ}\)C), then purified under a vacuum on membrane filters (0.45 \(\upmu\)m), and evaporated at 40 \({}^{\circ}\)C until dry in a Hei-VAP Precision (Heidolph, Schwabach, Germany) vacuum evaporator. The residue was dissolved in 15 mL of water with a 1% acetic acid addition. Honey sample solutions were applied in their entirety on conditioned cartridges with a C18 bed (Sep-Pak C18 500 mg, Waters, Milford, MA, USA). Polyphenolic compounds were eluted with 10 mL of methanol directly into a round-bottomed flask and evaporated at 40 \({}^{\circ}\)C until dry. The residue was dissolved in 2.5 mL MS grade methanol, filtered through nylon filters with a 0.22 \(\upmu\)m pore diameter (Biospace, Poznan, Poland), and subsequently analysed.