## 3 Results and Discussion
### Effects of Rice Stem Cell Extracts against Oxidative Damage on HFB  
To confirm the regenerative effect of HFB on N\({}_{2}\) u-DBD plasma-treated RSCE, HFB was first treated with 1 mM tert-butyl hydroperoxide (t-BHP) to artificially induce oxidative stress. Tert-butyl hydroperoxide (tBHP) is a well-known oxidative stress inducer used to induce cellular senescence [32; 33]. This substance depletes cellular antioxidant defense mechanisms, leading to lipid peroxidation [34; 35]. For this experiment, we used a human skin fibroblast (HFB) cell line. We used the various concentrations of callus extracts to observe the proliferation effects of HFB cells. To investigate the effects on the proliferation assay, cells were incubated with 10% FBS in DMEM. Various t-BHP concentrations (12.5, 25, 50, 100, 300, and 500 uM) were tested, and cell proliferation was evaluated (Supplementary Figure S3). As shown in Figure 5, plasma-treated RSCE was treated for each concentration (0.001, 0.01, 0.1, and 1 mg/mL), and the cell proliferation rate of HFB was compared. HFB cells were pretreated with RSCE that was plasma-treated for 24 h before t-BHP treatment. In Figure 5, the number of viable cells in HFB under t-BHP treatment increased the most using RSCE treated with a 0.1 mg/mL concentration of plasma for 3 min. Through the comparison of HFB viability using the concentration for plasma-treated RSCE in a state in which t-BHP was not treated, it was confirmed that 0.1 mg/mL was the optimal concentration (Supplementary Figure S4). The concentration of the extract from the 0.1 mg/mL group was shown to have higher effects than the other three groups (0.001, 0.01, and 1 mg/mL) after 24 h (Supplementary Figure S4), and it still consists of the effects until 48 h (data not shown). Plasma-treated RSCE at a concentration of 0.1 mg/mL showed a significant difference and increased the proliferation of HFB cells. RSCE at a concentration of 0.1 mg/mL treated for 1 min and 3 min was increased compared with RSCE without plasma treatment, and RSCE at a concentration of 0.1 mg/mL treated for 5 min showed a similar cell viability to that of the untreated control (Supplementary Figure S4). This trend showed a marked difference when treated with t-BHP oxidative stress. The 3 min plasma treated with 0.1 mg/mL RSCE showed an excellent ability to overcome the oxidative stress of t-BHP. In order to confirm the cell proliferation of plasma-treated rice stem cell extracts (RSCE), HFB was pretreated with plasma-treated RSCE at each concentration for 24 h. The plasma treatment lasted 1, 3, and 5 min, and nitrogen gas (1500 sccm) was used as the feeding gas. The RSCE was used in concentrations of 0.001, 0.01, 0.1, and 1 mg/mL. All measurements were made in three or more replicates. * \(p\) < 0.05; **_p_ < 0.01. \begin{table}
\begin{tabular}{c c c} \hline
**Retention Time (min)** & **Metabolites 1** & **Peak Height Changed 2** \\ \hline
34.805 & Fructose & 3 \(>\) 1 \(>\) C = 5 \\
36.690 & Galactopyranoside & 3 \(>\) 1 \(>\) C \(>\) 5 \\
47.830 & Melibose & 3 \(>\) C = 1 = 5 \\
49.325 & Glycoside & 3 \(>\) 1 \(>\) C \(>\) 5 \\
49.620 & Sucrose & 3 \(>\) 1 \(>\) C \(>\) 5 \\ \hline \end{tabular} 1 Candidate metabolites according to the retention time. 2 The difference in the height of the changed peaks in order from highest to lowest. C, untreated control; 1, 3, 5; each plasma treatment time.  
\end{table} 
Table 3: Candidate metabolites in rice stem cells. As a result of measuring the concentration of active species generated by plasma discharge, the concentration of NO\({}_{2}\) increased to about 3 \(\upmu\)L/L at 4 min of discharge and continued. NO\({}_{2}\) was dominant until 3 min of plasma discharge, but when plasma discharge was more than 3 min, the concentration of NO\({}_{2}\) was saturated and O\({}_{3}\) increased. Based on the results of offsetting the concentration of active species after 3 min of plasma discharge, it was speculated that NOx-type RNS including NO\({}_{2}\) played an important role in RSCE activity.