## 3 Results
### Antioxidant Capacity of Selected Enriched OPC Subfractions and Crude Extract In Vitro  
In addition to the cell-free TEAC assay, the antioxidant capacity of the test-substances and the crude extract was tested in vitro using a modified dichlorofluorescein diacetate (DCFH\({}_{2}\)-DA) assay. This assay provides indications of the antioxidant effect at the cellular level, whereas cell-free test systems such as the TEAC or FRAP (ferric-reducing antioxidant power) only provide information on the chemical reductive properties of the tested substances. Therefor DCFH\({}_{2}\)-DA is used as a fluorophore, which is only activated and hence reactive after cellular uptake. For the modification, the cells were pre-incubated with the test-substances for 1 h and subsequently treated with DCFH\({}_{2}\)-DA. The cells were ultimately treated with TBH to induce intracellular ROS [24]. A reduction in the TBH-induced relative fluorescence over time is an indication of antioxidant capacities due to intracellular ROS scavenging by the test compound. Results are described in comparison to the corresponding positive control (TBH incubation without pre-incubation), and the relative fluorescence intensity increases are shown for 15, 30, 45 and 60 min after the TBH was added (Figure 9). For SmSTri, SmSCin, SmSTet and SmSPen, the pre-incubation was performed for 1 h in a concentration range of 1-50 \(\upmu\)M.  
Figure 9: Results of (**1**) SmSTri, (**2**) SmSCin, (**3**) SmSTet and (**4**) SmSPen for the antioxidant potential via modified DCFH\({}_{2}\)-DA in vitro assay. Relative fluorescence intensity increases in HT29 cells after application of DCFH\({}_{2}\)-DA and 250 \(\upmu\)M tert-butyl hydroperoxide (TBH)—except for the negative control (1% DMSO). Pre-incubation for 1 h (except NC and positive control (PC)) with (**1**) SmSTri 1–50 \(\upmu\)M; (**2**) SmSCin 1–50 \(\upmu\)M; (**3**) SmSTet 1–50 \(\upmu\)M; (**4**) SmSPen 1–50 \(\upmu\)M. \(n=3\times 3\); statistical evaluation after [PERSON]’s test (\(p=95\%\)) via Student’s two-way \(t\)-test against the positive control with \(p\) as significance value (* \(p=95\%\), ** \(p=99\%\)). An antioxidant potential could not be proven for all tested compounds, as it was the case in the cell-free TEAC assay. Only SmSTri, SmSCin and SMSTet show antioxidant effects. SmSTri and SmSTet display concentration-dependent antioxidant effects over the time period of 60 min. For SmSTri (Figure 9 (1)), a significant effect occurs at the highest concentration of 50 uM at the time points of 30, 45 and 60 min. The clearest concentration dependency for this compound is after a short incubation period of 15 min with TBI. With time, the relative fluorescence of the PC and the test analytes become more and more similar, for the lower concentrations. SmSTet (see Figure 9 (3)) shows similar effects as SmSTri, whereby a significant antioxidant effect was observed at 50 uM and 15 min after TBI incubation. An overall reduction in the relative fluorescence intensity increase could be observed over the displayed time period for this compound. In addition, further significant effects occur at a concentration of 20 uM and 60 min as well as 1 uM and 5 uM after 10 min incubation with TBIH (Figure 9 (2)). Concentration-dependent effects are not as pronounced with SmSCin as with SmSTri and SmSTet. At the highest concentration of 50 uM, there is hardly any reduction in the relative fluorescence intensity increase. No significant antioxidative effects were observed for SmSPen (Figure 9 (4)) over the entire duration. For the two lowest concentrations, however, visible antioxidant effects occur over the entire period. The antioxidant potential of the crude extract was tested analog to SmSTri, SmSCin, SmSTet and SmSPen. Quercetin was included as a control substance, similar to the TEAC assay. Results for the crude extract are shown in Figure 10.  
The highest antioxidant properties of the crude extract was develop 15 min after ROS induction.