# Phytochemical Screening and Antioxidant Activity of Selected Estonian _Galium_ Species
## 3 Materials and Methods
### Chemicals  
Ultrapure water (\(\geq\)18 M\(\Omega\)cm) produced within the laboratory with a Milli-Q water purification system (Merck KGaA, Darmstadt, Germany) was used to prepare all aqueous solutions. Extraction solvent ethanol (96.7%) was obtained from Sigma-Aldrich (Munich, Germany). Acetonitrile (\(\geq\)99.9%), an HPLC eluent, and acetone (\(\geq\)99.9%), an extraction solvent, were purchased from Honeywell (Seelze, Germany). Formic acid (\(\geq\)99.0%) used in HPLC eluents was purchased from Fisher Chemical (Czech Republic). Total polyphenic content of the extracts was evaluated using the 2 M Folin-Ciocalteu reagent, purchased from Sigma-Aldrich (Switzerland), and water-free sodium carbonate, purchased from Sigma-Aldrich (Germany). Standard solutions used were prepared from gallic acid monohydrate (Sigma-Aldrich, China) and 96.7% ethanol (Sigma-Aldrich, Germany). For the detection of total  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Compound**} & **Rt,** & _Galium verum_ & _Galium verum_ & _Galium aparine_ & _Galium molligo_ \\  & **min** & **Blossoms,** & **Herb,** & **Herb,** & **Herb,** \\  & **\%** & **\%** & **\%** & **\%** & **\%** \\ \hline \(\alpha\)-Curcumene & 25.40 & \textless{}1.0 & 3.1 & - & - \\ \(\beta\)-Cubebene & 25.49 & 10.6 & \textless{}1.0 & - & - \\ Germancene D & 25.50 & \textless{}1.0 & 1.6 & - & 13.4 \\ \(\beta\)-Acoreenol & 25.64 & \textless{}1.0 & 1.2 & \textless{}1.0 & 2.6 \\ Aciphylene & 25.82 & - & 1.1 & - & - \\ Isobisabolene & 26.04 & 5.3 & 1.0 & \textless{}1.0 & 2.8 \\ Bornyl pentanoate & 26.24 & 1 & - & - & - \\ Dihydroactinidiolide & 26.67 & - & 2.5 & 8.5 & 10.2 \\ Caryophyllene oxide & 27.90 & - & 1.5 & - & 2.0 \\ Hexahydroseudioinone & 33.22 & - & \textless{}1.0 & 8.1 & 5.1 \\ \hline \hline \end{tabular}
\end{table}
Table 4: _Cont._flavonoid content, aluminium chloride, obtained from Fluka (Switzerland), was used. Standard solutions were prepared from quercetin (\(\geq\)99.0%, Lachema/Chemapol) and methanol (\(\geq\)99.9%, Honeywell, Charlotte, NC, USA). Total iridoid content was determined using the Trim-Hill reagent, prepared from water-free acetic acid (\(\geq\)99.0%, Sigma-Aldrich, Germany), 37% hydrochloric acid (Honeywell/Fluka, Austria), and copper sulfate pentahydrate (Sigma-Aldrich, Munich, Germany). Standard solutions were prepared using asperuloside purchased from MedChemExpress. Fluorescein sodium salt (\(\geq\)98.5%) and AAPH (2,2^-azobis(2-methyl-propionamidine) dihydrochloride, 97%) used in the antioxidants studies, were purchased from Fluka (Switzerland) and Sigma-Aldrich (Germany), respectively. Standard solutions were prepared from Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, 97%), purchased from Sigma-Aldrich (Germany). Standard compounds used for phytochemical quantification, asperuloside (\(\geq\)95%), neochlorogenic acid (\(\geq\)98%), chlorogenic acid (\(\geq\)95%), rutin (\(\geq\)95%), and quercetin (\(\geq\)95%), and internal standard, bicalutamide (\(\geq\)99.8%), were all purchased from Sigma-Aldrich (Germany).