## 3 Results and Discussion
### Effect of Alkaloid Extracts on TDH in PC-12 Cells Induced by OS with H\({}_{2}\)O\({}_{2}\)
Among the enzyme levels influenced by the alkaloid treatments, the ranking was PON1 > CAT > GPx > SOD (as depicted in Figure 5). The literature supports these findings. For instance, studies by [PERSON] et al. [92] and [PERSON] et al. [93] found that ND patients displayed substantially lower levels of PON1, SOD, CAT, and GPx compared to healthy individuals. [PERSON] et al. [94] reported the positive effects of berberine chloride, an isoquinoline alkaloid, on the antioxidant enzyme activities of rats exposed to the toxin diazinon. [PERSON] et al. [95] demonstrated that palmit, another isoquinoline alkaloid, significantly enhanced the activity of the SOD gene responsible for antioxidant defenses in a specific worm model. Additionally, [PERSON] et al. [96] studied the protective attributes of an alkaloid-rich extract from _Coperus rotundus_ L. (CR) against neurotoxicity in rats. They found that while exposure to the toxin esfenvalerate reduced brain PON1 levels, treatment with the _C. rotundus_ extract notably countered this toxicity by elevating PON1 levels. Our findings align with these studies, suggesting that our alkaloid extracts, abundant in isoquinoline alkaloids, offer protection against OS by bolstering the activities of crucial antioxidant enzymes, including PON1, SOD, CAT, and GPx. Figure 4: Levels of TOS, TAS, and OSI (H\({}_{2}\)O\({}_{2}\): Hydrogen peroxide, CAE: Chloroform alkaloid extract, MAE: Methanol alkaloid extract, MAE: Water alkaloid extract). The figures were presented as a mean \(\pm\) SD. [PERSON]’s test was used in the case of \(p\) < 0.05. \({}^{\#}\) Significantly different from the control (nz 3 for each bar). * Significantly different from the H\({}_{2}\)O\({}_{2}\) (n: 3 for each bar). Impact of Alkaloid Extracts on GCLC, HO-1, NRF2, NQO1, and KEAP1 Gene Expression Levels in PC-12 Cells Induced by OS with H\({}_{2}\)O\({}_{2}\)  
The NRF2-ARE signaling pathway plays a pivotal role in neurodegenerative diseases (NDs). The ARE is a recognized enhancer sequence (RTGACnmGC) found in the 5\({}^{\prime}\) regions of many genes responsible for phase II detoxification and antioxidant activities. In the cell's cytoplasm, NRF2, a protein, is anchored by KEAP1, an actin-binding protein. KEAP1, which operates as a Cul3-based E3 ligase, facilitates NRF2's polyubiquitin, making it a target for degradation by proteasomes. However, when exposed to oxidative stress (OS) or electrophilic agents that react with KEAP1, NRF2 becomes stabilized, accumulates in the nucleus, and pairs with small MAF proteins. This complex then binds to the ARE, prompting the expression of detoxification and antioxidant genes such as GCLC, HO-1, and NQO1 [97]. Various studies have highlighted that natural compounds, including alkaloids, can enhance the NRF2-mediated antioxidant pathway, which is essential for ND prevention [98]. In light of this, we evaluated the impact of alkaloid extracts on the gene expression of GCLC, HO-1, NRF2, NQO1, and KEAP1 in PC-12 cells subjected to OS via H\({}_{2}\)O\({}_{2}\) using qRT-PCR. Notably, in the H2O2 group, the expression of GCLC, HO-1, NRF2, and NQO1 genes diminished significantly compared to controls (\(p<0.05\)). Alkaloid extracts, based on their concentrations, markedly reversed this suppression (\(p<0.05\)). Meanwhile, H\({}_{2}\)O\({}_{2}\) exposure led to a notable rise in KEAP1 gene expression (\(p<0.05\)), which the alkaloid extracts managed to reduce, depending on their concentration (\(p<0.05\)). The effectiveness of the alkaloid concentrations ranked as 500 \(\mathrm{\SIUnitSymbolMicro m}\)/mL \(>\) 250 \(\mathrm{\SIUnitSymbolMicro m}\)/mL \(>\) 100 \(\mathrm{\SIUnitSymbolMicro m}\)/mL. The influence of the solvents depended on gene expression levels, with chloroform predominantly affecting NRF2 and KEAP1 and methanol affecting GCLC, HO-1, and NQO1. Among the affected genes, the sequence was NRF2 \(>\) NQO1 \(>\) KEAP1 \(>\) GCLC \(>\) HO-1 (illustrated in Figure 6). Corroborating our findings, [PERSON] et al. [99] and [PERSON] et al.