## 2 Materials and Methods
### General Metabolomics Procedure and UPLC-Orbitrap-Mass Spectrometry-Based Analysis  
Metabolite profiling was performed on Orbitrap Exploris 120 mass spectrometer coupled to the Vanquish Flex UPLC system (Thermo Fisher Scientific, San Jose, CA, USA) at the Biopolymer Research Center for Advanced Materials (Sejong University, Seoul, Republic of Korea). UV/Vis detector was conducted on a Vanquish Diode Array Detector FG of 210, 254, and 315 nm (Thermo Fisher Scientific, San Jose, CA, USA). The chromatographic method of the analysis was carried out on an ACQUITY UPLC BEH(r) C\({}_{18}\) column (2.1 \(\times\) 100 mm, 1.7 \(\upmu\)m, Waters, Milford, MA, USA). Thin-layer chromatography (TLC) was conducted us  
\begin{table}
\begin{tabular}{p{128.0 pt} p{128.0 pt} p{128.0 pt} p{128.0 pt}} \hline \hline
**Sample** & RL1 & RL2 & RL3 \\
**Code** & & & \\ \hline
**Sample** & KR391 & KR CheongHong & Bordeaux \\ \hline
**Plant Material** & & & \\ \hline \hline \end{tabular}
\end{table}
Table 1: Name, code, and the sample of _Raphanus sativus_ cultivars.  
ing precoated silica gel F254 plates and reverse-phase (RP)-C18 F254s plates (Merck). Spots on TLC were detected using UV and heating after dipping in anisaldehyde-sulfuric acid.  
Metabolite profiling of the aerial parts of _R. sativus_ extracts (RL1-3) was conducted using UPLC-Orbitrap-MS. The mobile phases consisted of 0.1% formic acid in distilled water (mobile phase A) and acetonitrile (mobile phase B). The gradient elution program was set as 1-95% B (0-16 min) and 100% B (16-18 min), followed by a re-equilibration at 1% B (18-20 min). The samples were also detected by UV/Vis spectra, and the chromatographic monitoring was performed at 210, 254, and 280 nm with a flow rate of 0.4 mL/min. The autosampler and column compartment temperatures were maintained at 4 \({}^{\circ}\)C and 40 \({}^{\circ}\)C, respectively. Mass spectrometry (MS) analysis utilized a full scan range of \(m/z\) 150-2000 in centroid mode with a resolution of 60,000. Key MS parameters included ion transfer tube temperature (320 \({}^{\circ}\)C), sheath gas (0 arb), auxiliary gas (0 arb), and spray voltage (3500 V). Data acquisition was managed using Xcalibur 4.6 software (Thermo Fisher Scientific). Raw data were processed with Thermo Scientific FreeStyle and Compound Discoverer 3.3, enabling compound identification by matching \(m/z\) values and MS/MS fragments against the Natural Product Atlas (npatlas) database [23]. The accuracy (\(\Delta\) ppm) of the data was calculated using the formula below, where \(\mathrm{mas}_{\mathrm{exp}}\) represents the experimental \(m/z\) and \(\mathrm{mas}_{\mathrm{calc}}\) corresponds to the theoretical \(m/z\) value from the molecular formulas in the literature [24].  
\[\Delta(\mathrm{ppm})=\frac{\left(\mathrm{mass}_{\mathrm{exp}}-\mathrm{mass}_{ \mathrm{calc}}\right)}{\mathrm{mass}_{\mathrm{exp}}}\times 10^{6}\]  
After data processing, the intensities of the identified metabolites were normalized against the peak area of quercetin, used as an internal standard (IS). SIMCA 18 (Umetrics, Inc., Umea, Sweden) was utilized for multivariate statistical analysis.  
Principal component analysis (PCA) was performed to verify the consistency and reliability of the analytical process before normalization. Quality control (QC) samples, prepared by pooling equal aliquots from all samples, were analyzed every tenth sample during the batch to ensure data quality. Following normalization, PCA was conducted to confirm clustering patterns among RL1, RL2, and RL3 groups.  
Statistical significance was assessed using f-tests to evaluate the homogeneity of variances, followed by _t_-tests to compare the means between RL1 and RL2, as well as between RL1 and RL3. A significance threshold of \(p\) < 0.05 was applied, and changes relative to RL1 were evaluated to determine significant increases or decreases.