## 2 Results
### Identification of Screened Antioxidants in the Methanol Extract of Marigold  
Potential antioxidant compounds were detected via offline DPPH-HPLC-Q-TOF/MS. The molecular weights of the four antioxidant compounds were determined by Q-TOF-MS/MS in negative ion mode, and their MS/MS information are shown in Table 2. Compound 2 was used as an example for mass spectrometry analysis. The EIC of the theoretical mass (\(m/z\) 317.0303, [M-H]-) of a reported compound in the TIC of the flower (Figure 2A) was determined. The result showed that Compound 2 appeared in high abundance (Rt 12.144 min), and its measured MS fragmentation negative mode was \(m/z\) 317.0303 (& ppm: 1.57) (Figure 2B). The MS/MS spectrum of this compound (Figure 2C) suggests that the first fragmentation pathway produced the fragment by the retro-Diels-Alder (RDA) reaction, and the fragments were \(m/z\) 166.9982 and 149.0239. The second fragmentation pathway involved the loss of the small molecules CO and H\({}_{2}\)O from the deprotonated skeleton, and the ions at \(m/z\) 139.0036 were formed by the neutral loss of CO moieties from the precursor ions at \(m/z\) 166.9982. Compound 2 was preliminarily identified as QG by analysing its mass spectra and references [32] (Figure 2D). Similarly, by analysing the MS spectra (Figure S1), peak 1, 3 and 4 were preliminarily identified as corresponding to quercetin-7-O-glucose (1), quercetin (3) and patuletin (4). Figure 1: Chromatograms of the methanol extract of marigold before and after reaction with DPPH radicals (**a**); chromatograms of the methanol extract of marigold with PG chromatogram before and after reaction with DPPH radicals (**b**). Quantitative Analysis of Quercetageetin-7-O-Glucoside, QG and Quercetin in Methanol Extracts of Marigolds  
Quantitative analyses of the main natural antioxidants, including quercetin-7-O-glucose (1), quercetin (2) and quercetin (3), were carried out with an external standard method. Calibration curves were constructed from peak areas as a function of compound concentrations. The three reference compounds were eluted at 6.615, 8.104 and 9.718 min, respectively, as seen in the chromatogram (Figure S2a). In the marigold extract sample, the three compounds were eluted at the same retention time and exhibited baseline separation from other components in the sample (Figure S2b). Calibration curves as well as correlation coefficients (R2) and linear ranges are listed in Table S2. The contents of quercetin-7-O-glucose (1), quercetin (2) and quercetin (3) were calculated, and the average contents were 31.02 \(\pm\) 0.96 \(\upmu\)g/mg, 65.18 \(\pm\) 2.08 ug/mg and 0.46 \(\pm\) 0.05 \(\upmu\)g/mg. The data showed that quercetin exhibited the highest content among the antioxidant components in marigold. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Peak No.** & **tR (min)** & **Name** & **Formula** & **[M-H]-(m/z)** & **Error** & **MS/MS Fragment Ions** \\ \hline
1 & 9.262 & Quercetageetin-7-O-glucose & C\({}_{21}\)H\({}_{20}\)O\({}_{13}\) & 479.0829 & 0.62 & 317.0304, 359.0403 \\
2 & 12.144 & Quercetagetin & C\({}_{15}\)H\({}_{10}\)O\({}_{8}\) & 317.0303 & 1.57 & 139.0036, 166.9982, 149.0239, 121.0294, 111.0086, 271.0247 \\
3 & 14.547 & Quercetin & C\({}_{15}\)H\({}_{10}\)O\({}_{7}\) & 301.0371 & 7.28 & 151.0039, 178.9998, 121.0296 \\
4 & 15.748 & Patuletin & C\({}_{16}\)H\({}_{20}\)O\({}_{8}\) & 331.0449 & \(-\)1.51 & 316.0216, 165.9896, 209.0081 \\ \hline \hline \end{tabular} Note: error (ppm) = (Measured \(m/z\) values—theoretical \(m/z\) values)/theoretical \(m/z\) values \(\times\) 10\({}^{6}\).  
\end{table} 
Table 2: Identified potential antioxidant compounds of the methanol extract of marigold. Figure 2: Screening of compound 2 by accurate-target means in the TIC (**A**), EIC (**B**) and MS/MS spectra (**C**) and the fragmentation pathway of compound 2 in the methanol extract of marigold flower (**D**).