###### Abstract  
Natural products with medicinal properties are among alternative therapies of interest due to their high body tolerance. We aimed to determine whether nonthermal gas plasma could enhance the medicinal value of _Flammulina velutipes_ mushrooms. Generated gas plasma was characterized by its emission spectrum in ambient air, pH, temperature, and H\({}_{2}\)O\({}_{2}\) and NO\({}_{x}\) concentrations after exposure for various periods. Phenolic and flavonoid contents in the extracts were measured using antioxidant assays and Fourier transform infrared and ultraviolet-visible spectroscopy. We analyzed the effects of the plasma-treated mushroom-derived extracts against breast carcinoma using the MCF7 and MDA-MB231 cell lines. The extracts significantly and concentration dependently inhibited the growth of breast cancer cells without inducing toxicity in normal MCF10A cells, and induced apoptosis via oxidative stress, evidenced by DNA damage (\(\gamma\)-H2 AX foci formation), and increased the population of MCF7 breast cancer cells arrested in the G2/M phase of the cell cycle. The extracts also induced mitochondrion-mediated apoptosis of MCF7 cells through cytochrome c release and caspase cleavage activity. The plasma improved the biological activity of mushrooms by increasing their phenolic compounds that prevented the growth of breast cancer cells in vitro.  
gas plasma; mushroom; anticancer; cell death +
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effects, including immunity enhancement and reduced blood cholesterol levels and pressure, as well as chemopre benefits. This is owing to its bioactive constituents [2]. Although extracts of this mushroom are significantly active against different breast cancer cell lines due to its antioxidant properties [3], it has not been comprehensively studied. The nutritional value of mushrooms is highly dependent on the cultivation method. The primary objective behind the cultivation strategy of any organic product is to boost its yield. Currently, an agricultural support system that helps increase production with minimal environmental side effects is required. Plasma has practical applications in the agricultural and food industries, and the contributory role of nonthermal plasma to agriculture has been established [4; 5; 6]. An increase in mushroom growth induced by nonthermal gas plasma (NTP) might be exploited to further enhance crop yield.  
Generally, reactive species and other plasma-induced components contribute to initiating growth mechanism or signaling direct plasma treatment. Plasma discharge can create neutral atoms and molecules, radicals, excited states, ions, and electrons, together with electrical energy [7]. These energetic electrons can generate radicals, charged species, excited states, and ultraviolet photons. With these unique properties, NTP sources can create a chemically productive environment. Without increasing the temperature, plasma generates an exclusive environment in which highly reactive plasma species can be delivered in a nondestructive and beneficial way to biological samples [8; 9]. Studies have highlighted the various pharmacological properties of _F. velutipes_ such as antimicrobial [10], anti-inflammatory [11], antioxidant [2], and immunomodulatory [12] and anticancer [2; 12] activities. Water-based extracts of _F. velutipes_ can inhibit growth and induce apoptosis in breast cancer cells [13]. Moreover, whole extracts or their purified components, such as polysaccharides and sterols, can affect cancer cell migration, inhibit growth, and exert immunomodulatory activities [14; 15; 16]. Due to the safety associated with plasma, exposing natural products such as _F. velutipes_ to gas plasma to enhance its phytochemical properties, as well as yield, is rapidly gaining popularity. This study aimed to determine the effectiveness of plasma-treated mushroom-derived extracts on breast cancer cells with safety considerations for their normal counterparts.