## 3 Results and Discussion
### Intracellular Reactive Oxygen Species (ROS) and Mitochondrial Superoxide Generations  
Our previous study reported that HnMc induces apoptosis in fungal cells via the generation of ROS and mitochondrial superoxide [23]. HnMc has a random coil structure in an aqueous solution, but when it binds to the fungal cell membrane, it forms an alpha-helical structure, damages the cell membrane, and enters the cell itself. Once inside the cell, it affects the outer membrane of the mitochondria and causes superoxide production because the outer membrane of the mitochondria is similar in composition to the bacterial cell membrane. In conclusion, it inhibited fungal growth by inducing apoptosis. Apoptosis is characterized by a progressive activation pathway that leads to specific biochemical and morphological events, such as ROS generation, imbalance of intracellular ions, cell shrinkage, destabilization of lipid asymmetry, chromatin condensation in the early stages, activation of caspases and endonucleases, formation of apoptotic bodies, and cell fragmentation in the late stages [24; 25].  
Total ROS generation was detected using DCF-DA, a fluorescent probe oxidized by ROS. The green fluorescence emission of the H2O2-treated fungus was 85.024% of the total, confirming that ROS detection was normal. Fungi treated with melittin and HnMc-WP2 were measured at 3.956% and 0.13%, respectively. However, in HnMc, HnMc-W, and HnMc-WP1, ROS-producing fungi were detected between 22 and 40% (Figure 5). To confirm the action of the peptides that entered the cell, superoxide production in the mitochondria was confirmed using MitoSOX Red fluorescence staining. As shown in Figure 6, fungi treated with H2O2 were not stained with MitoSOX Red, unlike DCF-DA, which showed that this dye is selectively oxidized by superoxide rather than ROS. Melittin and HnMc-WP2 produced little mitochondrial superoxide, whereas the other peptides produced high levels of superoxide. These results suggest that HnMc, HnMc-W, and HnMc-WP1 peptides inhibit fungal growth by entering cells and producing intracellular ROS through mitochondrial damage and that HnMc-WP2 acts on the fungal cell membrane as well as melittin.  
Figure 4: Membrane-permeable effects of peptides in _C. albicans_ CCARM 14007. After addition of SYTOX Green to peptide-treated fungal cells for 15 min, fungal cells were analyzed using flow cytometry (**A**) and fluorescent microscope (**B**). (**a**) control; (**b**) melittin; (**c**) HnMc; (**d**) HnMc-W; (**e**) HnMc-WP1; and (**f**) HnMc-WP2.  
Figure 5: ROS generation of peptides in _C. albicans_ CCARM 14007. Fungal cells were incubated without peptide (**a**) or with H\({}_{2}\)O\({}_{2}\) (**b**), melittin (**c**), HnMc (**d**), HnMc-W (**e**), HnMc-WP1 (**f**), and HnMc-WP2 (**g**) for 6 h; stained with DCF-DA; and analyzed under flow cytometry.  
Figure 6: Production of mitochondrial superoxide from _C. albicans_ CCARM 14007 treated with peptides. Fungal cells were incubated without peptide (**a**) or with H\({}_{2}\)O\({}_{2}\) (**b**), melittin (**c**), HnMc (**d**), HnMc-W (**e**), HnMc-WP1 (**f**), and HnMc-WP2 (**g**) for 6 h; stained with MitoSOX Red; and analyzed under flow cytometry.