## 3 Results and Discussion
### Biochemical Analyses
However, the amounts of this class of secondary metabolites were significantly higher in the hibiscus beer than in the control one, in which they were not present. The presence of anthocyanins in the beer was probably due to the hibiscus flowers, as they usually are not reported as main phenolic compounds of beer [41]. On the contrary, flavonoids are key components of this alcoholic beverage since they contribute to its astringency and bitterness [41; 42]. The total flavonoid contents (TFCs) were also assessed in all the samples, with their highest presence in the hibiscus flowers (73.91 mg CE/g DW) and hop cones (27.29 mg CE/g DW), and among the beverages, the hibiscus liquefur (741.26 ug/mL). Concerning the beer samples, the TFCs were significantly more abundant in the treated beer (360.66 ug CE/mL) than in the control one (201.37 ug CE/mL). Finally, the total polyphenol content (TPC) was investigated for all the samples. Both the plant materials presented suitable amounts of polyphenols, even if they were more abundant in the hibiscus flowers (114.57 mg GAE/g) than in the hop cones (99.45 mg GAE/g). The  
\begin{table}
\begin{tabular}{c c c c c c} \hline  & \multicolumn{2}{c}{_Hibiscus_} & \multicolumn{2}{c}{_HumulusInputus_} & & & \\ _rosa-sinensis_ **L** & **cv. Cascade L.** & **Cones** & **Control Beer** & **Hibiscus Beer** & **Hibiscus** & **Liqueur** \\ \hline Total chlorophylls & \(8.28\pm 1.69\) & \(671.77\pm 65.64\) & & & & \\ (ug/g DW) & 229.29 \(\pm\) 24.26 & \(161.17\pm 7.66\) & & & & \\ Total anthocyanins & 16.26 \(\pm\) 0.98 & nd & & & & \\ (mg/g DW) & & & nd b  & 2.76 \(\pm\) 0.26 a  & 134.21 \(\pm\) 5.24 \\ (ug CAE/mL) & & & 27.29 \(\pm\) 1.89 & & & \\ (mg CE/g DW) & 73.91 \(\pm\) 6.9 & 27.29 \(\pm\) 1.89 & & & \\ Total flavonoids & & & 201.37 \(\pm\) 5.83 b  & 360.66 \(\pm\) 4.72 a  & 741.26 \(\pm\) 5.83  & 741.26 \(\pm\) 5.83  \\ (mg GAE/g DW) & 114.57 \(\pm\) 4.89 & 99.45 \(\pm\) 5.68 & & & \\TPCs, as the anthocyanins, were highest in the hibiscus liqueuur (847.66 \(\upmu\)g GAE/mL), while the hibiscus beer presented a significantly higher amount than the control beer (436.26 \(>\) 350.31 \(\upmu\)g GAE/mL). Polypphenols are chemical compounds comprising several classes of compounds, including flavonoids and anthocyanins. In beer, they are mainly derived from the malt or the cereal used for the brewing process [41]. However, the type and the quality of the used raw materials can also significantly affect their content [42]. In the present work the addition of the hibiscus flowers to the beer resulted in an increase in these chemicals, which are of the utmost importance in the brewing process, as they can affect several characters of the beer such as the astringency, body and fullness, as well as the taste and aroma [41]. Moreover, polyphenols have a key role in the prevention of oxidation, since they are able to chelate free metals and neutralize free radicals with different mechanism of action--this could lead to an improvement of the product stability and to an increase in the shelf-life [43]. The radical scavenger activity of the samples was also assessed with a DPPH assay and expressed as a DPPH scavenging effect (\(\%\)). The _H. rosa sinensis_ plant samples showed higher activity than the hop cones, following the tendency of the antioxidant metabolites. The beverages with hibiscus, either the beer or the liqueur, exhibited strong scavenging effects of 74.71\(\%\) and 73.45\(\%\), respectively, while the control beer showed half the activity.