Abstract The western honey bee ( Apis mellifera) is increasingly exposed to environmental stressors that affect redox homeostasis, leading to imbalances in cellular functions. Natural bioactive compound-based nutritional strategies show promise in reducing oxidative stress while preserving redox signaling. In this study, we investigated the chemical composition, cytotoxicity, and redox-modulating effects of an aqueous extract of the edible mushroom Agaricus bisporus on the AmE-711 honey bee cell line. High-resolution Orbitrap LC–MS analysis revealed a chemically diverse extract comprising polyols, organic acids, amino acids, phosphorylated sugars, nucleotide derivatives, phenolic, and lipid-related compounds. Among the identified metabolites were mannitol, malic acid, citric acid, glutamic acid, and uridine diphosphate N-acetylglucosamine, providing a biochemical basis for potential metabolic and redox-related activity. Cell viability assays demonstrated that A. bisporus extract exhibited no significant cytotoxicity under the experimental conditions. Electron paramagnetic resonance (EPR) spectroscopy with the TEMPONE spin probe showed that untreated cells exhibited only minimal signal reduction (4.20%), while treatment with the extract alone caused a moderate decrease (12.08%), indicating the absence of reductive stress. Oxidative stress induced by hydrogen peroxide resulted in a pronounced TEMPONE signal reduction (37.88%), whereas co-treatment with the A. bisporus extract substantially attenuated this effect, lowering the signal reduction to 15.34%. These findings suggest that the aqueous A. bisporus extract may help preserve basal redox activity while attenuating peroxide-induced oxidative stress in AmE-711 honey bee cells. Rather than acting as a potent radical scavenger, the extract appears to function as a mild redox modulator or stabilizer under the tested conditions, which may be beneficial for honey bee cellular redox balance. These results support further investigation of physiologically appropriate A. bisporus-based dietary supplements for mitigating oxidative stress in apicultural systems. 1. Introduction Through its pollination activity, the western honey bee ( Apis mellifera) is essential to both agricultural productivity and ecosystem stability [ 1]. However, a number of environmental stressors, such as pesticide exposure, pathogens, nutritional deficiencies, and habitat degradation, have caused significant declines in honey bee populations in recent decades [ 2]. The disruption of cellular redox homeostasis, leading to oxidative stress that compromises immune competence, metabolic function, and overall bee colony resilience, is a common physiological consequence of these stressors [ 3, 4]. As inevitable byproducts of aerobic metabolism, reactive oxygen species (ROS) play crucial signaling roles in insects, including regulation of immunity, development, and stress responses [ 5]. Tightly regulated redox signaling is essential for preserving cellular homeostasis in honey bees [ 6]. However, an excessive build-up of ROS can overpower the natural antioxidant defenses, causing oxidative damage to proteins, lipids, and nucleic acids [ 7]. On the other hand, excessive ROS suppression by high-dose antioxidant supplementation may cause reductive stress and disrupt physiological signaling pathways [ 8, 9]. Therefore, it is becoming more widely acknowledged that a more physiologically appropriate approach to promoting insect health is to use nutritional interventions that stabilize rather than eradicate redox activity. The “white button” mushroom Agaricus bisporus is one of the most extensively grown edible fungi in the world and is known to contain a wide range of bioactive substances, such as polyols, organic acids, amino acids, polysaccharides, and phenolic derivatives [ 20, 21]. Its remarkable nutritional, medicinal, and cosmetic values are reviewed by Usman et al. [ 22]. This mushroom is considered a healthy food due to multiple bioactive properties, including antioxidant and immunomodulatory effects [ 22, 23]. The potential of A. bisporus in animal nutrition and veterinary medicine is less studied, but available studies indicate favorable effects on production traits and animal health [ 24, 25, 26, 27, 28]. Although A. bisporus has been linked to immunomodulatory, anti-inflammatory, and antioxidant effects in mammalian systems [ 28, 29, 30, 31], little is known about its cellular mechanisms of action and possible advantages in insects. Among insects, investigations of A. bisporus extract were conducted only on honey bees and a honey bee cell line, and all showed its beneficial effects. Better survival and reduced levels of Nosema ceranae infection [ 32, 33], up-regulation of immune-related genes [ 32], and mitigation of pesticide-induced oxidative stress were recorded in caged bees supplemented with the A. bisporus extract [ 33]. Furthermore, antigenotoxic/genoprotective effects of the same extract have been revealed by the Comet assay on honey bee cells [ 34]. Finally, in a field experiment, an extract of A. brasiliensis (a relative species of A. bisporus) improved the strength of and contributed to the increase in honey and pollen stores in supplemented bee colonies [ 35]. Although previous studies demonstrated beneficial effects of A. bisporus extracts in honey bees, including reduced Nosema ceranae infection, improved survival, mitigation of pesticide-induced oxidative stress, and antigenotoxic activity, the underlying cellular redox mechanisms remain insufficiently characterized. In particular, no previous study has combined high-resolution LC–MS metabolite profiling with direct EPR-based monitoring of intracellular redox dynamics in honey bee-derived cells. Furthermore, the effects of A. bisporus metabolites on the redox status of the AmE-711 honey bee cell line have not yet been investigated. The AmE-711 cell line, derived from A. mellifera embryonic tissues, represents a well-established in vitro model for investigating cellular responses in honey bees under controlled experimental conditions. Although embryonic cells do not fully reproduce the complexity of adult bee physiology, they retain conserved metabolic and redox-regulatory pathways relevant to oxidative stress responses [ 41]. Their use enables reproducible assessment of cytotoxicity, oxidative imbalance, and cellular redox modulation while minimizing biological variability inherent to whole-organism studies. An aqueous A. bisporus extract was studied with the aims: (i) to characterize its metabolite profile using high-resolution LC-MS; (ii) to assess its cytotoxicity in AmE-711 honey bee cell line; and (iii) to examine its effects on cellular redox dynamics under basal and oxidative stress conditions using electron paramagnetic resonance (EPR) spectroscopy. By integrating high-resolution qualitative LC–MS metabolite profiling with EPR-based monitoring of intracellular redox activity in AmE-711 cells, this study provides new insights into the potential mechanisms underlying the redox-modulating effects of A. bisporus extract. 4. Conclusions An aqueous extract from the edible mushroom species A. bisporus has been shown in this study to have a balanced and biologically compatible redox-modulating activity on honey bee cells. High-resolution LC-MS analysis revealed that the extract is primarily composed of primary metabolites, including polyols, organic acids, amino acids, phosphorylated sugars, and nucleotide derivatives, with minor but biologically significant phenolic and lipid-related compounds, which together provide a plausible biochemical foundation for the observed cellular responses. The cell viability tests showed that the extract exhibited no significant cytotoxicity under the experimental conditions used. The results of EPR spectroscopy tests revealed that the extract did not cause excessive redox suppression under basal conditions. Under conditions of hydrogen peroxide-induced oxidative stress, the extract effectively attenuated redox imbalance and restored intracellular redox activity toward a controlled physiological range. In fact, A. bisporus extract is unlikely to exert its protective effects through high concentrations of classical antioxidants, but rather through the collective and synergistic actions of low-molecular-weight metabolites that support cellular metabolism, stabilize redox cofactors, and indirectly enhance endogenous antioxidant and stress-response systems. The potential advantages of A. bisporus extracts for the entire organism, immune function, and resistance to environmental stressors such as pathogens and agrochemicals should be considered when analyzing the effects of dietary supplementation with these extracts. The strategy presented in this paper has the potential to be helpful in creating long-term dietary plans that promote the health of honey bees in contemporary apiculture. At the end, it should be noted that this study was carried out on the AmE-711 cell line, derived from embryonic tissues [ 102], so further studies in adult bees and under field conditions are needed. Supplementary Materials The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules31122011/s1, Figure S1: Cell viability (%) of AmE-711 honey bee cells in the negative control (PBS), A. bisporus extract-treated (5 mg/mL), and positive control (100 µM H 2O 2) groups. Data are expressed as mean ± SE ( n = 3). One-way ANOVA revealed no statistically significant differences (n.s.) among groups (F(2,6) = 2.58, p = 0.155). Author Contributions Conceptualization, Đ.N. and U.G. (Uroš Glavinić), M.M. and Z.S.; methodology, Đ.N., U.G. (Uroš Glavinić), U.G. (Uroš Gašić), and J.S.; software, M.M.; validation, Đ.N., U.G. (Uroš Gašić), M.M. and Z.S.; formal analysis, Đ.N., U.G. (Uroš Gašić), and M.R.; investigation, Đ.N., M.M., U.G. (Uroš Glavinić), U.G. (Uroš Gašić) and J.S.; resources, U.G. (Uroš Glavinić), M.M. and Z.S.; data curation, Đ.N., U.G. (Uroš Gašić) and M.R.; writing—original draft preparation, Đ.N., U.G. (Uroš Gašić) and J.S.; writing—review and editing, Đ.N., J.S., U.G. (Uroš Glavinić), Z.S. and M.M.; visualization, Đ. N., U.G. (Uroš Gašić) and M.R.; supervision, Z.S. and M.M.; project administration, U.G. (Uroš Glavinić), Z.S. and M.M.; funding acquisition, U.G. (Uroš Glavinić), M.M. and Z.S. All authors have read and agreed to the published version of the manuscript. Funding This research was funded by the Science Fund of the Republic of Serbia, project Waste2Protect Bees No. 5455, led by Uroš Glavinić; Ministry of Science, Technological Development and Innovation of the Republic of Serbia, Contract numbers: 451-03-33/2026-03/200007, and 451-03-33/2026-03/200146, 451-03-34/2026-03/200146 for the project led by Miloš Mojović, and 451-03-34/2026-03/200143, for the project led by Zoran Stanimirović. Data Availability Statement All data underlying the results are available as part of the article and no additional source data are required. Conflicts of Interest The authors declare no conflicts of interest. References Papa, G.; Maier, R.; Durazzo, A.; Lucarini, M.; Karabagias, I.K.; Plutino, M.; Bianchetto, E.; Aromolo, R.; Pignatti, G.; Ambrogio, A.; et al. The honey bee Apis mellifera: An insect at the interface between human and ecosystem health. Biology 2022, 11, 233. [] [ CrossRef] Stanimirovic, Z.; Glavinic, U.; Ristanic, M.; Aleksic, N.; Jovanovic, N.; Vejnovic, B.; Stevanovic, J. Looking for the causes of and solutions to the issue of honey bee colony losses. Acta Vet. 2019, 69, 1–31. [] [ CrossRef] Tahir, F.; Goblirsch, M.; Adamczyk, J.; Karim, S.; Alburaki, M. Honey bee Apis mellifera L. responses to oxidative stress induced by pharmacological and pesticidal compounds. Front. Bee Sci. 2023, 1, 1275862. [] [ CrossRef] Tlak Gajger, I.; Cvetkovikj, A. Antioxidant potential of pollen polyphenols in mitigating environmental stress in honeybees ( Apis mellifera). Antioxidants 2025, 14, 1086. [] [ CrossRef] González-Tokman, D.; Villada-Bedoya, S.; Hernández, A.; Montoya, B. Antioxidants, oxidative stress and reactive oxygen species in insects exposed to heat. Curr. Res. Insect Sci. 2025, 7, 100114. [] [ CrossRef] Mackei, M.; Huber, F.; Oláh, B.; Neogrády, Z.; Mátis, G. Redox metabolic disruptions in the honey bee brain following acute exposure to the pyrethroid deltamethrin. Sci. Rep. 2025, 15, 28322. [] [ CrossRef] Manful, C.F.; Fordjour, E.; Subramaniam, D.; Sey, A.A.; Abbey, L.; Thomas, R. Antioxidants and reactive oxygen species: Shaping human health and disease outcomes. Int. J. Mol. Sci. 2025, 26, 7520. [] [ CrossRef] [ PubMed] Chandimali, N.; Bak, S.G.; Park, E.H.; Lim, H.-J.; Won, Y.-S.; Kim, E.-K.; Park, S.-I.; Lee, S.J. Free radicals and their impact on health and antioxidant defenses: A review. Cell Death Discov. 2025, 11, 19. [] [ CrossRef] Zhang, S.; Wang, N.; Gao, Z.; Gao, J.; Wang, X.; Xie, H.; Wang, C.-Y.; Zhang, S. Reductive stress: The key pathway in metabolic disorders induced by overnutrition. J. Adv. Res. 2025, 77, 569–584. [] [ CrossRef] Tauber, J.P.; Collins, W.R.; Schwarz, R.S.; Chen, Y.; Grubbs, K.; Huang, Q.; Lopez, D.; Peterson, R.; Evans, J.D. Natural product medicines for honey bees: Perspective and protocols. Insects 2019, 10, 356. [] [ CrossRef] Lamontagne-Drolet, M.; Samson-Robert, O.; Giovenazzo, P.; Fournier, V. The impacts of two protein supplements on commercial honey bee ( Apis mellifera L.) colonies. J. Apic. Res. 2019, 58, 800–813. [] [ CrossRef] Jovanovic, N.M.; Glavinic, U.; Delic, B.; Vejnovic, B.; Aleksic, N.; Mladjan, V.; Stanimirovic, Z. Plant-based supplement containing B-complex vitamins can improve bee health and increase colony performance. Prev. Vet. Med. 2021, 190, 105322. [] [ CrossRef] Brown, A.F.; Wiedmer, S.; Retschnig, G.; Neumann, P. Feeding with plant powders increases longevity and body weight of Western honeybee workers ( Apis mellifera). Apidologie 2024, 55, 54. [] [ CrossRef] Garrido, P.M.; Porrini, M.P.; Alberoni, D.; Baffoni, L.; Scott, D.; Mifsud, D.; Eguaras, M.J.; Di Gioia, D. Beneficial bacteria and plant extracts promote honey bee health and reduce Nosema ceranae infection. Probiotics Antimicro. 2024, 16, 259–274. [] [ CrossRef] [ PubMed] Danmek, K.; Wu, M.-C.; Kliathin, K.; Ng, H.L.; Hongsibsong, S.; Ghosh, S.; Jung, C.; Chuttong, B. The potential of mulberry leaf protein concentrate as a supplementary feed on the health and lifespan of honey bees ( Apis mellifera L.). J. Ecol. Environ. 2024, 48, 452–461. [] [ CrossRef] Ewert, A.M.; McMenamin, A.; Adjaye, D.; Rainey, V.; Ricigliano, V. Microalgae functional feed additives strengthen immunity and increase longevity in honey bees. J. Invertebr. Pathol. 2025, 211, 108352. [] [ CrossRef] Jovanovic, N.M.; Glavinic, U.; Ristanic, M.; Vejnovic, B.; Ilic, T.; Stevanovic, J.; Stanimirovic, Z. Effects of plant-based supplement on oxidative stress of honey bees ( Apis mellifera) infected with Nosema ceranae. Animals 2023, 13, 3543. [] [ CrossRef] Glavinić, U.; Džogović, D.; Jelisić, S.; Ristanić, M.; Zorc, M.; Aleksić, N.; Stanimirović, Z. Oxidative status of honey bees infected with Nosema ceranae microsporidium and supplemented with Agaricus bisporus mushroom extract. Vet. Glas. 2023, 77, 35–50. [] [ CrossRef] Glavinić, U.; Nakarada, Đ.; Stevanović, J.; Gašić, U.; Ristanić, M.; Mojović, M.; Stanimirović, Z. Chemical composition and antioxidant activity of Prokupac grape pomace extract: Implications for redox modulation in honey bee cells. Antioxidants 2025, 14, 751. [] [ CrossRef] [ PubMed] Bhambri, A.; Srivastava, M.; Mahale, V.G.; Mahale, S.; Karn, S.K. Mushrooms as potential sources of active metabolites and medicines. Front. Microbiol. 2022, 13, 837266. [] [ CrossRef] Paulauskienė, A.; Tarasevičienė, Ž.; Šileikienė, D.; Česonienė, L. The quality of ecologically and conventionally grown white and brown Agaricus bisporus mushrooms. Sustainability 2020, 12, 6187. [] [ CrossRef] Usman, M.; Murtaza, G.; Ditta, A. Nutritional, medicinal, and cosmetic value of bioactive compounds in button mushroom ( Agaricus bisporus): A review. Appl. Sci. 2021, 11, 5943. [] [ CrossRef] Atila, F.; Owaid, M.N.; Shariati, M.A. The nutritional and medical benefits of Agaricus bisporus: A review. J. Microbiol. Biotechnol. Food Sci. 2017, 7, 281–286. [] [ CrossRef] Giannenas, I.; Tontis, D.; Tsalie, E.; Chronis, E.F.; Doukas, D.; Kyriazakis, I. Influence of dietary mushroom Agaricus bisporus on intestinal morphology and microflora composition in broiler chickens. Res. Vet. Sci. 2020, 89, 78–84. [] [ CrossRef] Giannenas, I.; Tsalie, E.; Chronis, E.F.; Mavridis, S.; Tontis, D.; Kyriazakis, I. Consumption of Agaricus bisporus mushroom affects the performance, intestinal microbiota composition and morphology, and antioxidant status of turkey poults. Anim. Feed Sci. Technol. 2011, 165, 218–229. [] [ CrossRef] Mršić, G.; Špoljarić, D.; Valpotić, H.; Balenović, M.; Kozačinski, L.; Špoljarić, I.; Valpotić, I.; Savić, V.; Srečec, S.; Popović, M. Immunomodulatory effects of white button Agaricus bisporus supplementation in broiler chickens. Vet. Stanica 2011, 42, 431–439. [] Shamsi, S.; Seidavi, A.; Rahati, M.; G Nieto, J.Á. Edible mushroom powder ( Agaricus bisporus) and flavophospholipol improve performance and blood parameters of broilers. Rev. Colomb. Cienc. Pec. 2015, 28, 291–302. [] [ CrossRef] Špoljarić, D.; Srečec, S.; Paro, M.K.; Čop, M.J.; Mršić, G.; Šimpraga, B.; Sokolović, M.; Crnjac, J.; Špiranec, K.; Popović, M. The effects of feed supplemented with Agaricus bisporus on health and performance of fattening broilers. Vet. Arh. 2015, 85, 309–322. [] Jiang, N.; Xu, S.; Li, C. Research progress on pharmacological activity of Agaricus bisporus: A review. Food Biosci. 2025, 73, 107763. [] [ CrossRef] Michalska, A.; Sierocka, M.; Drzewiecka, B.; Świeca, M. Antioxidant and anti-inflammatory properties of mushroom-based food additives and food fortified with them-Current status and future perspectives. Antioxidants 2025, 14, 519. [] [ CrossRef] Muszyńska, B.; Kała, K.; Rojowski, J.; Grzywacz, A.; Opoka, W. Composition and biological properties of Agaricus bisporus fruiting bodies—A review. Pol. J. Food Nutr. Sci. 2017, 67, 173–181. [] [ CrossRef] Glavinic, U.; Rajkovic, M.; Vunduk, J.; Vejnovic, B.; Stevanovic, J.; Milenkovic, I.; Stanimirovic, Z. Effects of Agaricus bisporus mushroom extract on honey bees infected with Nosema ceranae. Insects 2021, 12, 915. [] [ CrossRef] [ PubMed] Jelisić, S.; Stanimirović, Z.; Ristanić, M.; Nakarada, Đ.; Mojović, M.; Bošnjaković, D.; Glavinić, U. The potential of Agaricus bisporus in mitigating pesticide-induced oxidative stress in honey bees infected with Nosema ceranae. Life 2024, 14, 1498. [] [ CrossRef] Rajkovic, M.; Stanimirovic, Z.; Stevanovic, J.; Ristanic, M.; Vejnovic, B.; Goblirsch, M.; Glavinic, U. Evaluation of genotoxic and genoprotective effects of Agaricus bisporus extract on AmE-711 honey bee cell line in the Comet assay. J. Apic. Res. 2024, 63, 769–777. [] [ CrossRef] Stevanovic, J.; Stanimirovic, Z.; Simeunovic, P.; Lakic, N.; Radovic, I.; Sokovic, M.; Griensven, L.J. The effect of Agaricus brasiliensis extract supplementation on honey bee colonies. An. Acad. Bras. Ciênc. 2018, 90, 219–229. [] [ CrossRef] Mayirnao, H.-S.; Jangir, P.; Sharma, K.; Kaur, S.; Sharma, Y.P.; Kapoor, R. Nutrient and antioxidant profile of four species of wild mushrooms from cold-desert with implications for human dietary and supplement use. Food Chem. Adv. 2025, 7, 101023. [] [ CrossRef] Podkowa, A.; Kryczyk-Poprawa, A.; Opoka, W.; Muszyńska, B. Culinary–medicinal mushrooms: A review of organic compounds and bioelements with antioxidant activity. Eur. Food Res. Technol. 2021, 247, 513–533. [] [ CrossRef] Silva, M.; Lageiro, M.; Ramos, A.C.; Reboredo, F.H.; Gonçalves, E.M. Cultivated mushrooms: A comparative study of antioxidant activity and phenolic content. Biol. Life Sci. Forum 2024, 40, 13. [] [ CrossRef] DeBerardinis, R.J.; Lum, J.J.; Hatzivassiliou, G.; Thompson, C.B. The biology of cancer: Metabolic reprogramming fuels cell growth and proliferation. Cell Metab. 2008, 7, 11–20. [] [ CrossRef] [ PubMed] DeBerardinis, R.J.; Thompson, C.B. Cellular metabolism and disease: What do metabolic outliers teach us? Cell 2012, 148, 1132–1144. [] [ CrossRef] [ PubMed] Goblirsch, M.; Adamczyk, J.J. Using the honey bee ( Apis mellifera L.) cell line AmE-711 to evaluate insecticide toxicity. Environ. Toxicol. Chem. 2023, 42, 88–99. [] [ CrossRef] Pabasara, G.V.S.; Fernando, M.D.M.; Abeysekera, W.K.S.M.; Liyanapathirana, L.V.C. Comparative study on the therapeutic potential of aqueous extracts from commercially cultivated Agaricus bisporusLentinula edodes in Sri Lanka: Antioxidant, anticancer, antidiabetic, and antibacterial properties. BMC Complement. Med. 2025, 25, 233. [] [ CrossRef] Vunduk, J.; Kozarski, M.; Klaus, A.; Jadranin, M.; Pezo, L.; Todorović, N. Preventing mislabeling of organic white button mushrooms ( Agaricus bisporus) combining NMR-based foodomics, statistical, and machine learning approach. Food Res. Int. 2024, 198, 115366. [] [ CrossRef] [ PubMed] Vunduk, J.; Djekic, I.; Petrović, P.; Tomašević, I.; Kozarski, M.; Despotović, S.; Nikšić, M.; Klaus, A. Challenging the difference between white and brown Agaricus bisporus mushrooms: Science behind consumers choice. Brit. Food J. 2018, 120, 1381–1394. [] [ CrossRef] Reis, G.C.L.; Dala-Paula, B.M.; Tavano, O.L.; Guidi, L.R.; Godoy, H.T.; Gloria, M.B.A. In vitro digestion of spermidine and amino acids in fresh and processed Agaricus bisporus mushroom. Food. Res. Int. 2020, 137, 109616. [] [ CrossRef] [ PubMed] Ruthes, A.C.; Rattmann, Y.D.; Malquevicz-Paiva, S.M.; Carbonero, E.R.; Córdova, M.M.; Baggio, C.H.; Santos, A.R.; Gorin, P.A.; Iacomini, M. Agaricus bisporus fucogalactan: Structural characterization and pharmacological approaches. Carbohydr. Polym. 2013, 92, 184–191. [] [ CrossRef] Patyshakuliyeva, A.; Jurak, E.; Kohler, A.; Baker, A.; Battaglia, E.; de Bruijn, W.; Burton, K.S.; Challen, M.P.; Coutinho, P.M.; Eastwood, D.C.; et al. Carbohydrate utilization and metabolism is highly differentiated in Agaricus bisporus. BMC Genom. 2013, 14, 663. [] [ CrossRef] Jankov, M.; Léguillier, V.; Gašić, U.; Anba-Mondoloni, J.; Ristivojević, M.K.; Radoičić, A.; Dimkić, I.; Ristivojević, P.; Vidic, J. Antibacterial activities of Agaricus bisporus extracts and their synergistic effects with the antistaphylococcal drug AFN-1252. Foods 2024, 13, 1715. [] [ CrossRef] [ PubMed] Petrović, J.; Glamočlija, J.; Milinčić, D.D.; Doroški, A.; Lević, S.; Stanojević, S.P.; Kostić, A.Ž.; Popović Minić, D.A.; Vidović, B.B.; Plećić, A.; et al. Comparative chemical analysis and bioactive properties of aqueous and glucan-rich extracts of three widely appreciated mushrooms: Agaricus bisporus (J.E.Lange) Imbach, Laetiporus sulphureus (Bull.) Murill and Agrocybe aegerita (V. Brig.) Vizzini. Pharmaceuticals 2024, 17, 1153. [] [ CrossRef] [ PubMed] Gąsecka, M.; Magdziak, Z.; Siwulski, M.; Mleczek, M. Profile of phenolic and organic acids, antioxidant properties and ergosterol content in cultivated and wild growing species of Agaricus. Eur. Food Res. Technol. 2018, 244, 259–268. [] [ CrossRef] Yang, W.; Wu, Y.; Hu, Q.; Mariga, A.M.; Pei, F. Ultrahigh-pressure liquid chromatography-quadrupole-time-of-flight mass spectrometry-based metabolomics reveal the mechanism of methyl jasmonate in delaying the deterioration of Agaricus bisporus. J. Agr. Food Chem. 2019, 67, 8773–8782. [] [ CrossRef] [ PubMed] Nowak, A.; Piotrowska, M.; Przydacz, K.; Czyżowska, A.; Rajkowska, K.; Dybka-Stępień, K.; Koziróg, A.; Otlewska, A.; Budryn, G.; Kołczyk, A. Non-volatile bioactive properties of mushroom extracts ( Agaricus bisporusPleurotus ostreatus). Molecules 2026, 31, 1397. [] [ CrossRef] Jennemann, R.; Bauer, B.L.; Bertalanffy, H.; Geyer, R.; Gschwind, R.M.; Selmer, T.; Wiegandt, H. Novel glycoinositolphosphosphingolipids, basidiolipids, from Agaricus. Eur. J. Biochem. 1999, 259, 331–338. [] [ CrossRef] Wadman, M.W.; van Zadelhoff, G.; Hamberg, M.; Visser, T.; Veldink, G.A.; Vliegenthart, J.F.G. Conversion of linoleic acid into novel oxylipins by the mushroom Agaricus bisporus. Lipids 2005, 40, 1163–1170. [] [ CrossRef] Patel, T.K.; Williamson, J.D. Mannitol in plants, fungi, and plant–fungal interactions. Trends Plant Sci. 2016, 21, 486–497. [] [ CrossRef] [ PubMed] Zhao, X.; Yu, C.; Zhao, Y.; Liu, S.; Wang, H.; Wang, C.; Guo, L.; Chen, M. Changes in mannitol content, regulation of genes involved in mannitol metabolism, and the protective effect of mannitol on Volvariella volvacea at low temperature. BioMed Res. Int. 2019, 2019, 1493721. [] [ CrossRef] van Iersel, G.; van Brenk, B.; Bleichrodt, R.-J. Adaptive osmoregulation in successive flushes of Agaricus bisporus by free amino acids and mannitol. Fungal Biol. 2025, 129, 101688. [] [ CrossRef] [ PubMed] André, P.; Villain, F. Free radical scavenging properties of mannitol and its role as a constituent of hyaluronic acid fillers: A literature review. Int. J. Cosmet. Sci. 2017, 39, 355–360. [] [ CrossRef] Shen, B.; Jensen, R.G.; Bohnert, H.J. Mannitol protects against oxidation by hydroxyl radicals. Plant Physiol. 1997, 115, 527–532. [] [ CrossRef] Hou, L.; Huang, C.; Wu, X.; Zhang, J.; Zhao, M. Nitric oxide negatively regulates the rapid formation of Pleurotus ostreatus Primordia by inhibiting the mitochondrial aco gene. J. Fungi 2022, 8, 1055. [] [ CrossRef] Son, S.Y.; Park, Y.J.; Jung, E.S.; Singh, D.; Lee, Y.W.; Kim, J.-G.; Lee, C.H. Integrated metabolomics and transcriptomics unravel the metabolic pathway variations for different sized beech mushrooms. Int. J. Mol. Sci. 2019, 20, 6007. [] [ CrossRef] [ PubMed] Chandel, N.S. Evolution of mitochondria as signaling organelles. Cell Metab. 2015, 22, 204–206. [] [ CrossRef] [ PubMed] Murphy, M.P. How mitochondria produce reactive oxygen species. Biochem. J. 2009, 417, 1–13. [] [ CrossRef] [ PubMed] Newsholme, P.; Procopio, J.; Lima, M.M.R.; Pithon-Curi, T.C.; Curi, R. Glutamine and glutamate--their central role in cell metabolism and function. Cell Biochem. Funct. 2003, 21, 1–9. [] [ CrossRef] Gamarra, Y.; Santiago, F.C.; Molina-López, J.; Castaño, J.; Herrera-Quintana, L.; Domínguez, Á.; Planells, E. Pyroglutamic acidosis by glutathione regeneration blockage in critical patients with septic shock. Crit. Care 2019, 23, 162. [] [ CrossRef] Rohadi, A.; Lazim, A.M.; Hasbullah, S.A. The antioxidant effect of derivatives pyroglutamic lactam. AIP Conf. Proc. 2013, 1571, 801–805. [] [ CrossRef] Foulquier, E.; Pompeo, F.; Byrne, D.; Fierobe, H.-P.; Galinier, A. Uridine diphosphate N-acetylglucosamine orchestrates the interaction of GlmR with either YvcJ or GlmS in Bacillus subtilis. Sci. Rep. 2020, 10, 15938. [] [ CrossRef] Gharehzadehshirazi, A.; Amini, A.; Rezaei, N. Hyper IgE syndromes: A clinical approach. Clin. Immunol. 2022, 237, 108988. [] [ CrossRef] Chang, Y.H.; Weng, C.L.; Lin, K.I. O-GlcNAcylation and its role in the immune system. J. Biomed. Sci. 2020, 27, 57. [] [ CrossRef] Kiddane, A.T.; Kim, G.D. Anticancer and immunomodulatory effects of polysaccharides. Nutr. Cancer 2021, 73, 2219–2231. [] [ CrossRef] Barreto-Bergter, E.; Figueiredo, R.T. Fungal glycans and the innate immune recognition. Front. Cell. Infect. Microbiol. 2014, 4, 145. [] [ CrossRef] Kim, J.-E.; Takanche, J.-S.; Yun, B.-S.; Yi, H.-K. Anti-inflammatory character of Phelligridin D modulates periodontal regeneration in lipopolysaccharide-induced human periodontal ligament cells. J. Periodontal Res. 2018, 53, 816–824. [] [ CrossRef] Li, Y.; Zhou, Y.; Wu, J.; Li, J.; Yao, H. Phelligridin D from Inonotus obliquus attenuates oxidative stress and accumulation of ECM in mesangial cells under high glucose via activating Nrf2. J. Nat. Med. Tokyo 2021, 75, 1021–1029. [] [ CrossRef] Adepoju, F.O.; Duru, K.C.; Li, E.; Kovaleva, E.G.; Tsurkan, M.V. Pharmacological potential of betulin as a multitarget compound. Biomolecules 2023, 13, 1105. [] [ CrossRef] Haque, E.; Mahmud, Z.; Hasan, A.K.M.M.; Sardar, R.; Kabir, L.; Haque, T. Betulin-3-caffeate and amyrin from the stem bark of Barringtonia acutangula (L). Biores. Commun. 2015, 1, 121–123. [] Gowda, S.G.B.; Tsukui, T.; Fuda, H.; Minami, Y.; Gowda, D.; Chiba, H.; Hui, S.P. Docosahexaenoic acid esters of hydroxy fatty acid is a novel activator of NRF2. Int. J. Mol. Sci. 2021, 22, 7598. [] [ CrossRef] Truong, V.-L.; Bae, Y.-J.; Rarison, R.H.G.; Bang, J.-H.; Park, S.-Y.; Jeong, W.-S. Anti-inflammatory and antioxidant activities of lipophilic fraction from Liriope platyphylla seeds using network pharmacology, molecular docking, and in vitro experiments. Int. J. Mol. Sci. 2023, 24, 14958. [] [ CrossRef] Kozarski, M.; Klaus, A.; Jakovljevic, D.; Todorovic, N.; Vunduk, J.; Petrović, P.; Niksic, M.; Vrvic, M.M.; van Griensven, L. Antioxidants of edible mushrooms. Molecules 2015, 20, 19489–19525. [] [ CrossRef] [ PubMed] Ramos, M.; Burgos, N.; Barnard, A.; Evans, G.; Preece, J.; Graz, M.; Ruthes, A.C.; Jiménez-Quero, A.; Martínez-Abad, A.; Vilaplana, F.; et al. Agaricus bisporus and its by-products as a source of valuable extracts and bioactive compounds. Food Chem. 2019, 292, 176–187. [] [ CrossRef] Blumfield, M.; Abbott, K.; Duve, E.; Cassettari, T.; Marshall, S.; Fayet-Moore, F. Examining the health effects and bioactive components in Agaricus bisporus mushrooms: A scoping review. J. Nutr. Biochem. 2020, 84, 108453. [] [ CrossRef] [ PubMed] Babić, N.; Peyrot, F. Molecular probes for evaluation of oxidative stress by in vivo epr spectroscopy and imaging: State-of-the-art and limitations. Magnetochemistry 2019, 5, 13. [] [ CrossRef] Bačić, G.; Pavićević, A.; Peyrot, F. In vivo evaluation of different alterations of redox status by studying pharmacokinetics of nitroxides using magnetic resonance techniques. Redox Biol. 2016, 8, 226–242. [] [ CrossRef] Dudonné, S.; Vitrac, X.; Coutière, P.; Woillez, M.; Mérillon, J.M. Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J. Agric. Food Chem. 2009, 57, 1768–1774. [] [ CrossRef] Dai, J.; Mumper, R.J. Plant phenolics: Extraction, analysis and their antioxidant and anticancer properties. Molecules 2010, 15, 7313–7352. [] [ CrossRef] Abdelshafy, A.M.; Belwal, T.; Liang, Z.; Wang, L.; Li, D.; Luo, Z.; Li, L. A comprehensive review on phenolic compounds from edible mushrooms: Occurrence, biological activity, application and future prospective. Crit. Rev. Food Sci. 2022, 62, 6204–6224. [] [ CrossRef] [ PubMed] Nakarada, Đ.; Marković, S.; Popović, M.; Dimitrijević, M.; Rakić, A.; Mojović, M. Redox properties of grape wine skin extracts from the Šumadija region: An electron paramagnetic resonance study. Hosp. Pharmacol. Int. Multidiscip. J. 2021, 8, 1004–1013. [] [ CrossRef] Rockenbach, I.I.; Gonzaga, L.V.; Rizelio, V.M.; Gonçalves, A.E.D.S.S.; Genovese, M.I.; Fett, R. Phenolic compounds and antioxidant activity of seed and skin extracts of red grape ( Vitis viniferaVitis labrusca) pomace from Brazilian winemaking. Food Res. Int. 2011, 44, 897–901. [] [ CrossRef] Barros, L.; Venturini, B.A.; Baptista, P.; Estevinho, L.M.; Ferreira, I.C.F.R. Chemical composition and biological properties of portuguese wild mushrooms: A comprehensive study. J. Agr. Food Chem. 2008, 56, 3856–3862. [] [ CrossRef] [ PubMed] Ferreira, I.C.F.R.; Barros, L.; Abreu, R.M.V. Antioxidants in wild mushrooms. Curr. Med. Chem. 2009, 16, 1543–1560. [] [ CrossRef] Li, S.; Fasipe, B.; Laher, I. Potential harms of supplementation with high doses of antioxidants in athletes. J. Exerc. Sci. Fit. 2022, 20, 269–275. [] [ CrossRef] Sies, H.; Jones, D.P. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat. Rev. Mol. Cell Biol. 2020, 21, 363–383. [] [ CrossRef] Taric, E.; Glavinic, U.; Vejnovic, B.; Stanojkovic, A.; Aleksic, N.; Dimitrijevic, V.; Stanimirovic, Z. Oxidative stress, endoparasite prevalence and social immunity in bee colonies kept traditionally vs. those kept for commercial purposes. Insects 2020, 11, 266. [] [ CrossRef] Simone-Finstrom, M.; Li-Byarlay, H.; Huang, M.H.; Strand, M.K.; Rueppell, O.; Tarpy, D.R. Migratory management and environmental conditions affect lifespan and oxidative stress in honey bees. Sci. Rep. 2016, 6, 32023. [] [ CrossRef] [ PubMed] Zikic, B.; Aleksic, N.; Ristanic, M.; Glavinic, U.; Vejnovic, B.; Krnjaic, I.; Stanimirovic, Z. Anti- Varroa efficiency of coumaphos and its influence on oxidative stress and survival of honey bees. Acta Vet. 2020, 70, 355–373. [] [ CrossRef] Balieira, K.V.; Mazzo, M.; Bizerra, P.F.; Guimarães, A.R.; Nicodemo, D.; Mingatto, F.E. Imidacloprid-induced oxidative stress in honey bees and the antioxidant action of caffeine. Apidologie 2018, 49, 562–572. [] [ CrossRef] Chakrabarti, P.; Carlson, E.A.; Lucas, H.M.; Melathopoulos, A.P.; Sagili, R.R. Field rates of Sivanto™ (flupyradifurone) and Transform ପ୍ପ (sulfoxaflor) increase oxidative stress and induce apoptosis in honey bees ( Apis mellifera L.). PLoS ONE 2020, 15, e0233033. [] [ CrossRef] Glavinic, U.; Blagojevic, J.; Ristanic, M.; Stevanovic, J.; Lakic, N.; Mirilovic, M.; Stanimirovic, Z. Use of thymol in Nosema ceranae control and health improvement of infected honey bees. Insects 2022, 13, 574. [] [ CrossRef] [ PubMed] Kunat-Budzyńska, M.; Łabuć, E.; Ptaszyńska, A.A. Changes in enzymatic activity and oxidative stress in honeybees kept in the apiary and laboratory conditions during the course of nosemosis. PLoS ONE 2025, 20, e0317384. [] [ CrossRef] Paris, L.; Roussel, M.; Pereira, B.; Delbac, F.; Diogon, M. Disruption of oxidative balance in the gut of the western honeybee Apis mellifera exposed to the intracellular parasite Nosema ceranae and to the insecticide fipronil. Microb. Biotechnol. 2017, 10, 1702–1717. [] [ CrossRef] Orčić, S.; Nikolić, T.; Purać, J.; Šikoparija, B.; Blagojević, D.P.; Vukašinović, E.; Plavša, N.; Stevanović, J.; Kojić, D. Seasonal variation in the activity of selected antioxidant enzymes and malondialdehyde level in worker honey bees. Entomol. Exp. Appl. 2017, 165, 120–128. [] [ CrossRef] Kojić, D.K.; Purać, J.S.; Nikolić, T.V.; Orčić, S.M.; Vujanović, D.; Ilijević, K.; Vukašinović, E.L.; Blagojević, D.P. Oxidative stress and the activity of antioxidative defense enzymes in overwintering honey bees. Entomol. Gen. 2019, 39, 33–44. [] [ CrossRef] Goblirsch, M.J.; Spivak, M.S.; Kurtti, T.J. A cell line resource derived from honey bee ( Apis mellifera) embryonic tissues. PLoS ONE 2013, 8, e69831. [] [ CrossRef] [ PubMed] Stojković, D.; Gašić, U.; Uba, A.I.; Zengin, G.; Rajaković, M.; Stevanović, M.; Drakulić, D. Chemical profiling of Anthriscus cerefolium (L.) Hoffm., biological potential of the herbal extract, molecular modeling and KEGG pathway analysis. Fitoterapia 2024, 177, 106115. [] [ CrossRef] [ PubMed] Phillips, H.J. Dye exclusion tests for cell viability. In Tissue Culture: Methods and Applications; Kruse, P.F., Patterson, M.K., Eds.; Academic Press: Cambridge, MA, USA, 1973; pp. 406–408. [] Mojović, M.D.; Spasojević, I.; Vuletić, M.M.; Vučinić, Ž.B.; Bačić, G. An EPR spin-probe and spin-trap study of the free radicals produced by plant plasma membranes. J. Serb. Chem. Soc. 2005, 70, 177–186. [] [ CrossRef] Nakarada, Đ.; Pejin, B.; Tommonaro, G.; Mojović, M. Liposomal integration method for assessing antioxidative activity of water insoluble compounds towards biologically relevant free radicals: Example of avarol. J. Liposome Res. 2020, 30, 218–226. [] [ CrossRef] The representative EPR spectrum of TEMPONE. The representative EPR spectrum of TEMPONE. Effect of A. bisporus extract (AB) on TEMPONE oxidation in AmE-711 honey bee cells under basal and oxidative stress conditions. Data are presented as mean ± SE ( n = 3 independent experiments). Significance brackets (independent t-test, unadjusted p-value): * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical comparison was performed using one-way ANOVA followed by Tukey’s post hoc test. Effect of A. bisporus extract (AB) on TEMPONE oxidation in AmE-711 honey bee cells under basal and oxidative stress conditions. Data are presented as mean ± SE ( n = 3 independent experiments). Significance brackets (independent t-test, unadjusted p-value): * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical comparison was performed using one-way ANOVA followed by Tukey’s post hoc test. LC-MS data on metabolites identified in Agaricus bisporus extract. LC-MS data on metabolites identified in Agaricus bisporus extract. No Compound Name tR, min Molecular Formula, [M–H] −Calculated Mass, m/ zExact Mass, m/ zΔ mDa MS 2 Fragments, (% Base Peak) 1 Hexane-1,2,3,4,5,6-hexol 0.50 C 6H 13O 6−181.07189 181.07246 −0.57 59.01405(81), 71.01412(56), 85.02985(18), 89.02474(100), 101.02481(77), 181.07245(60) 2 Aspartic acid 0.51 C 4H 6NO 4−132.03020 132.03077 −0.57 88.04073(86), 95.02545(18), 113.03613(81), 114.02015(100), 132.03076(57) 3 Tetrose 0.52 C 4H 7O 4−119.03502 119.03563 −0.61 59.01407(93), 71.01427(35), 74.02512(78), 75.02862(62), 101.02441(21), 119.03564(100) 4 Deoxyhexose 0.52 C 6H 11O 5−163.06120 163.06192 −0.72 59.01406(73), 85.02994(53), 101.02490(34), 113.02499(10), 131.03543(9), 163.0619(100) 5 Hexose 0.52 C 6H 11O 6−179.05619 179.05685 −0.66 89.02477(81), 99.00914(35), 131.03560(31), 161.04614(65), 179.05685(100) 6 Glutamic acid 0.53 C 5H 8NO 4−146.04596 146.04650 −0.54 102.05645(100), 128.03589(54), 146.04649(43) 7 Threonine 0.53 C 4H 8NO 3−118.05100 118.05142 −0.42 72.00938(4), 74.02502(100), 118.05149(29) 8 Tetrahydroxypentanoic acid 0.54 C 5H 9O 6−165.04046 165.04100 −0.54 59.01407(15), 75.00906(100), 99.00917(12), 129.0199(14), 147.03053(9), 165.04111(61) 9 Glucose phosphate 0.55 C 6H 12O 9P −259.02247 259.02302 −0.55 78.95934(78), 96.96997(100), 138.98085(13), 241.01292(6), 259.02301(10) 10 Malic acid 0.55 C 4H 5O 5−133.01420 133.01483 −0.63 71.01413(38), 89.02479(7), 115.00418(100), 133.01483(47) 11 Fumaric acid 0.56 C 4H 3O 4−115.00370 115.00419 −0.49 71.01411(100), 115.00419(17) 12 N-(1-Deoxy-D-mannitol-1-yl)-L-glutamic acid 0.56 C 11H 20NO 9−310.11445 310.11540 −0.95 128.03580(100) 13 Pyroglutamic acid 0.57 C 5H 6NO 3−128.03532 128.03584 −0.52 128.03583(100) 14 Glycerol phosphate 0.57 C 3H 8O 6P −171.00646 171.00712 −0.66 78.95934(100), 96.96999(15), 171.00711(15) 15 N-Acetylglucosamine phosphate 0.58 C 8H 15NO 9P −300.04909 300.04990 −0.81 78.95931(100), 96.96993(82), 118.05138(33), 138.98077(5), 199.00186(4), 300.04990(6) 16 Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) 0.58 C 17H 26N 3O 17P 2−606.07435 606.07622 −1.87 78.95933(93), 96.96993(33), 158.92595(71), 176.93655(37), 272.95801(100), 282.03943(73), 362.00555(20), 384.98541(91), 402.99573(20) 17 Methylcitric acid 0.59 C 7H 9O 7−205.03538 205.03575 −0.38 71.05051(14), 87.00906(28), 99.04551(26), 101.02478(26), 125.02486(100), 145.01480(20) 18 Uridine monophosphate (5′-UMP) 0.60 C 9H 12N 2O 9P −323.02866 323.02895 −0.29 78.95933(100), 96.96996(84), 111.02045(18), 211.00206(15) 19 Aconitic acid 0.60 C 6H 5O 6−173.00916 173.00975 −0.59 85.02982(42), 111.00922(100), 129.01987(9) 20 2-Furoic acid 0.60 C 5H 3O 3−111.00880 111.00938 −0.58 67.01921(49), 111.00937(100) 21 Citric acid 0.60 C 6H 7O 7191.01973 191.02040 −0.67 85.02983(34), 87.00908(50), 111.00921(100), 129.01985(8), 173.00999(2), 191.02039(8) 22 Succinic acid 0.67 C 4H 5O 4−117.01930 117.01985 −0.55 73.02979(100), 99.0092(10), 117.01984(41) 23 Azelaic acid 7.07 C 9H1 5O 4−187.09758 187.09832 −0.73 97.06628(5), 125.09771(100), 169.08774(4), 187.0983(41) 24 Phelligridin D 7.83 C 20H 11O 8−379.04592 379.04736 −1.44 229.01508(16), 269.01013(100), 307.06235(17), 335.05756(36), 351.05225(16), 379.04736(70) 25 Phelligridin C 8.21 C 20H 11O 7−363.05108 363.05220 −1.12 217.01526(22), 269.01038(19), 307.06207(17), 319.06281(21), 335.05804(17), 363.05225(100) 26 Dihydroxyoctadecadienoic acid 8.84 C 18H 31O 4−311.22285 311.22495 −2.10 249.22385(8), 293.21329(100), 311.22495(41) 27 Betulin-3-caffeate 11.89 C 39H 55O 5−603.40552 603.40724 −1.72 161.02512(2), 603.40723(100) 28 Hydroxytetracosanoic acid 12.44 C 24H 47O 3−383.35307 383.35415 −1.08 337.34888(65), 365.34149(2), 383.35443(100) 29 Hydroxydocosanoic acid 13.88 C 22H 43O 3−355.32177 355.32278 −1.01 309.31738(64), 337.31339(2), 355.32278(100) Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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Metabolic Redox Modulation by Agaricus bisporus Aqueous Extract in Honey Bee Cells
