## 4 Discussion  
Natural products, such as functional foods, have gained popularity as they provide alternatives to conventional cancer therapies, which are often accompanied by side effects and drug resistance. Mushrooms are rich in phytometabolic and bioactive constituents, and thus comprise an excellent alternative therapy that has health-promoting and tumor preventive effects [32]. Possible roles of various mushroom species in cancer treatment have been investigated [33; 34], while others involving the optimization and improvisation of their constituents have already been directed towards anticancer therapeutics. In this regard, both direct and indirect treatment with NTP increases the yield and quality of plants including mushrooms, and improves other aspects of agriculture [35; 36]. Here, we applied an air plasma jet for 30 and 60 s from an optimized device to enhance the yield and properties of _F. veluipes_, and then assessed characteristics and amounts of metabolic constituents such as phenols and flavonoids determined by FTIR and UV-Vis. Higher peak intensities in the UV-Vis spectra of extracts of plasma-treated mushrooms, indicated higher concentrations of constituents. Higher levels of total phenols, flavonoids, and antioxidant activities indicated that plasma enhanced the properties of mushroom extracts and might further improve their functions or applications such as cancer cell attenuation. Natural products exhibit anticancer activity primarily via selective cytotoxicity toward cancer cells. The cell viability results indicated that the mushroom extracts could selectively affect the metabolic growth of cancer cells at specific doses, while normal mammary breast cells remained largely unharmed. Furthermore, this effect was significantly higher in MCF-7s than in MDA-MB-231 breast cancer cells. Water extracts of _F. veluipes_ can inhibit both MCF-7 and MDA-MB-231 cells at different IC\({}_{50}\) doses [13]. Water extracts of _F. velutipes_ decrease the anticancer activity more prominently in MDA-MB-231 than MCF7 cells. However, we found that ethanol extracts of were highly cytotoxic to MCF-7 cancer cells. This might have been due to extract solvents differently affecting cancer cell subtypes. Differences in intrinsic molecular signaling patterns within subtypes of cancer and susceptibility towards certain bioactive compounds might also be involved [37]. Further investigation should provide more clarity into this aspect. We also found that reduced cell viability was associated with cell death and changes in the cell cycle. Consistent with these findings, more MCF7 than normal cells died, indicating that the mushroom extract effectively induced cell death in breast cancer as compared with normal mammary cells. The Annexin-V/PI analysis revealed that mushroom extracts prepared from plasma-treated mushrooms could induce cell death by apoptosis (Figure 5), due to changes in cell death markers. Furthermore, apoptotic cell death was associated with cell cycle arrest, as MCF7 cells were induced into the G2/M phase, while the S-phase also seemed to be affected by the 30 s extract (200 \(\mu\)g mL\({}^{-1}\)) in MCF7 cells (Figure 6). At similar concentrations, control (untreated) groups also had the intrinsic ability to alter cell cycle phases. Several mushrooms contain high amounts of antioxidants such as polyphenol, and vital compounds that exert anticancer effects that might be realized through mechanisms similar to those of some drugs, for instance, Nrf inhibitors. Being rich in antioxidants, extracts of mushrooms can prevent tissue damage and apoptosis by scavenging ROS [38]. However, some mushroom extracts can induce ROS and subsequent apoptosis as anticancer effects [39]. We found that ROS levels were dose-dependently enhanced in groups incubated with mushroom extracts. Natural product-derived inhibitors of key pathways can induce ROS activity that can result in cell death [40; 41; 42]. However, cytotoxicity was maximal in the cells incubated with 30 s extracts, indicating ROS independent mechanisms.