## 3 Results and Discussion  
### Total Polyphenols, Flavonoids and Antioxidant Assays  
The outcomes of the test conducted are listed in Tables 3 and 4. Considering that the methanol extract has been proven to be the most effective in obtaining an extract with good antioxidant activity, all extracts were prepared using this solvent. In both DPPH (2,2-Diphenyl-1-picrylhydrazyl) and ABTS (2,2\({}^{\prime}\)-azino-bis(3-ethylbenzothazoline-6-sulfonic acid)) assays, the strongest radical scavenging ability was found in full bloom (I-II) samples with results of 62.19 to 171.40 mg TE/g and from 160.97 to 363.58 mg TE/g, respectively. The higher radical scavenging activities on DPPH or ABTS assays of the investigated samples could be justified by the high concentrations of polyphenols in the same extracts. Similar to radical scavenging assays, using the CUPRAC and FRAP assays, the best activities were observed in the same extracts. Also, in this case, the obtained results from radical scavenging and reducing power assays could be explained by the higher concentration of phenolic compounds. The antioxidant activity of the extracts was in fact consistent with their content of flavonoids and phenolic compounds, ranging from 46.71 to 60.62 mg RE/g and from 37.91 to 68.16 mg GAE/g, respectively. Comparing the obtained results with those of other authors, the resulting TPC and TFC were similar to those reported by [PERSON] et al. (2015) and by [PERSON] et al. (2016), but lower than those reported by [PERSON] et al. (2017). Nevertheless, the TPC and TFC values of \(H\). _perforatum_ L. are affected by a number of variables, including the extraction solvent, the altitude of selected habitats, etc. (2018). In addition, spectrophotometric methods are not sufficient to accurately quantify total polyphenols and flavonoids because they do not distinguish between target compounds and other possible interfering compounds occurring in a complex matrix such as a plant extract. Therefore, the same extracts were investigated in the contents of individual polyphenols using the HPLC-MS/MS system.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Compound**} & **Precursor Ion** & **Product Ion** & **Fragmentor** & **Collision Energy** & **Retention Time** & **Polarity** \\  & **(_m/z_)** & **(_m/z_)** & **(V)** & **(V)** & **(Rt, min)** & **Positive** \\ \hline \multirow{2}{*}{Melatonin} & 233.11 & 174.2 * & 78 & 12 & 11.16 & positive \\  & 233.11 & 159.1 & 78 & 28 & 11.16 & positive \\ \hline \hline \end{tabular}
* The product ion was used for quantification, the other to confirm the analyte.  
\end{table}
Table 2: HPLC-MS/MS acquisition parameters optimized for melatonin.  
\begin{table}
\begin{tabular}{c c c} \hline \hline  & **Total Phenolics** & **Total Flavonoids** \\
**Samples** & **Content** & **Content** \\  & **(mg GAE/g)** & **(mg RT/g)** \\ \hline \hline \(H.\)_perforatum_ & & \\ I & 68.16 \(\pm\) 1.85 \({}^{\text{a}}\) & 51.82 \(\pm\) 0.66 \({}^{\text{b}}\) \\ \(H.\)_perforatum_ & & \\ II & 65.63 \(\pm\) 2.19 \({}^{\text{a}}\) & 60.62 \(\pm\) 0.82 \({}^{\text{a}}\) \\ \(H.\)_perforatum_ & & \\ III & 37.91 \(\pm\) 0.13 \({}^{\text{b}}\) & 46.71 \(\pm\) 0.42 \({}^{\text{c}}\) \\ \hline \hline \end{tabular}  
* Values are reported as the mean \(\pm\) S.D. of three parallel measurements. TPC: total phenolic content; TFC: total flavonoid content; GAE: gallic acid equivalent; RE: rutin equivalent. Different letters indicate significant differences between the tested extracts (\(p\) < 0.05).  
\end{table}
Table 3: Total phenolic and flavonoid content.