## 3 Results and Discussion
### Screening for Hop Oil Bioactive Components by HPTLC Hyphenations  
The individual components of the commercial hop oils responsible for various bioactive effects were detected using in situ assays after separation by HPTLC. For this reason, two HPTLC methods were developed using \(n\)-hexane as the mobile phase for the separation of highly non-polar compounds, and chloroform was used for the separation of the other compounds. In the HPTLC-DPPH\(\bullet\) assay, no active zones were revealed, which indicated that the oils had weak antioxidant activity, as observed in the EPR tests. In this study, two HPTLC-EDA methods were used to detect hop oil compounds with potential anti-obesity activity, namely HPTLC-\(\alpha\)-glucosidase and HPTLC-lipase assays. All four zones (at \(hk_{\mathrm{F}}\) 45, 64, 73, and 87) separated with \(n\)-hexane and visualised with anisaldehyde possessed \(\alpha\)-glucosidase inhibition activity (Figure 3a-c). However, upon employing the generally established method for the \(\alpha\)-glucosidase assay, which used the 2-naphthyl-\(\alpha\)-D-glucopyranoside substrate and Fast Blue B chromogenic reagent, a strange dark colour appeared in the middle of the zone at \(hk_{\mathrm{F}}\) 64 and only the bright ring \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Hop Oil Variety**} & **TREAC \({}^{\ast}\)** & **GAEAC \({}^{\ast}\)** & **AAEAC \({}^{\ast}\)** \\  & **[\(\mu\)mol TR/100 mL Sample]** & **[\(\mu\)mol GA/100 mL Sample]** & **[\(\mu\)mol AA/100 mL Sample]** \\  & **(\(\pm\)SD)** & **(\(\pm\)SD)** & **(\(\pm\)SD)** \\ \hline Amarillo & 27.51 (\(\pm\)0.50) \({}^{\mathrm{c}}\) & 12.02 (\(\pm\)0.20) \({}^{\mathrm{c}}\) & 28.55 (\(\pm\)0.47) \({}^{\mathrm{c}}\) \\ \hline Azacca & 39.90 (\(\pm\)0.91) \({}^{\mathrm{b}}\) & 17.11 (\(\pm\)0.38) \({}^{\mathrm{b}}\) & 40.33 (\(\pm\)0.87) \({}^{\mathrm{b}}\) \\ \hline Cascade & 0.00 \({}^{\mathrm{e}}\) & 0.00 \({}^{\mathrm{e}}\) & 0.00 \({}^{\mathrm{e}}\) \\ \hline Chinook & 38.81 (\(\pm\)2.37) \({}^{\mathrm{b}}\) & 16.66 (\(\pm\)0.97) \({}^{\mathrm{b}}\) & 39.30 (\(\pm\)2.26) \({}^{\mathrm{b}}\) \\ \hline Centennial & 3.31(\(\pm\)0.63) \({}^{\mathrm{d}}\) & 2.08 (\(\pm\)0.24) \({}^{\mathrm{d}}\) & 5.33 (\(\pm\)0.29) \({}^{\mathrm{d}}\) \\ \hline Saaz & 0.00 \({}^{\mathrm{e}}\) & 0.00 \({}^{\mathrm{e}}\) & 0.00 \({}^{\mathrm{e}}\) \\ \hline Ahhhroma & 44.74 (\(\pm\)1.95) \({}^{\mathrm{a}}\) & 19.10 (\(\pm\)0.80) \({}^{\mathrm{a}}\) & 44.99 (\(\pm\)1.97) \({}^{\mathrm{a}}\) \\ \hline \hline \end{tabular}  
* TREAC, Trolox equivalent of antioxidant capacity; GAEAC, gallic acid equivalent of antioxidant capacity; AAEAC, ascorbic acid equivalent antioxidant capacity.  
\end{table} 
Table 3: Results of quantification of antioxidant capacity of 7 commercial hop oils, as established by EPR spectroscopy, against three different free radical scavengers, i.e., Trolox (TR), gallic acid (GA), and ascorbic acid (AA) (n = 3). Different letters indicate significantly different values within a column (\(p\) < 0.05). Figure 2: The effect of different volumes of hop oil on the EPR spectra of DPPH\(\bullet\) for commercial hop oil sample no. 7 (Ahhhroma) characterized by the highest Trolox equivalent antioxidant capacity (TREAC) values (see Table 3). implied inhibitory action of the present compound (s) (Figure 3b). This strange staining effect may have resulted from an interaction of the analyte with a chromogenic reagent. For this reason, elimination of the reagent was achieved by replacing the substrate with 4-methylumbelliferyl-\(\alpha\)-D-glucopyranoside (Figure 3c). Providing a fluorescent signal, this improved the assay and confirmed the enzyme inhibitory effect of the compounds in all four zones, although none of them inhibited the lipase enzyme. Moreover, two out of four zones (at \(hR_{\mathrm{F}}\) 73 and 87) also showed weak antibacterial activity against _A. fischeri_ (Figure 3d).