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Its survival within the gut microenvironment depends on metabolic pathways such as glycolysis, where glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays a central role, alongside interactions with nitric oxide (NO) derived from host defenses and surrounding microbiota. The emergence of metronidazole resistance necessitates exploration of alternative therapeutic strategies.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Objective<\/jats:title>\n                    <jats:p>\n                      To evaluate the in vitro effects of metronidazole, metformin, and\n                      <jats:italic>Allium cepa<\/jats:italic>\n                      (onion) extract on\n                      <jats:italic>Blastocystis<\/jats:italic>\n                      sp. growth, and GAPDH activity and NO levels within the xenic culture medium.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Twenty-one\n                      <jats:italic>Blastocystis<\/jats:italic>\n                      -positive stool samples were cultured in Jones\u2019 medium under xenic conditions. Cultures were treated with graded concentrations of metronidazole, metformin, and onion extract. Parasite counts were assessed microscopically, while GAPDH activity and NO levels were quantified using gas chromatography\u2013mass spectrometry (GC\u2013MS)-based analysis.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      All tested agents significantly reduced\n                      <jats:italic>Blastocystis<\/jats:italic>\n                      counts compared with untreated controls (\n                      <jats:italic>p<\/jats:italic>\n                      \u2009&lt;\u20090.05), with metronidazole demonstrating the greatest antiparasitic effect. Significant reductions in NO levels and GAPDH activity were observed across all treatment groups (\n                      <jats:italic>p<\/jats:italic>\n                      \u2009&lt;\u20090.001). Metformin (1000\u00a0\u00b5g\/mL) showed the strongest inhibition of GAPDH activity, whereas metronidazole (500\u00a0\u00b5g\/mL) resulted in the lowest NO levels. Correlation analysis revealed a significant negative association between NO and GAPDH in metronidazole-treated cultures, while a positive correlation was observed with metformin.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>\n                      Metronidazole, metformin, and onion extract exhibit significant anti-\n                      <jats:italic>Blastocystis<\/jats:italic>\n                      activity, accompanied by modulation of key metabolic and nitrosative pathways within the microbial milieu supporting the growth of\n                      <jats:italic>Blastocystis<\/jats:italic>\n                      sp. Targeting GAPDH and NO-related mechanisms represents a promising therapeutic approach, particularly in the context of emerging drug resistance and co-colonization with other gut microbiota.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12906-026-05605-x","type":"journal-article","created":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T04:54:14Z","timestamp":1791780854000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Evaluating the effects of metronidazole, metformin, and onion extract on GAPDH activity and nitric oxide production in Blastocystis sp. cultures: a biochemical and therapeutic approach"],"prefix":"10.1186","volume":"26","author":[{"given":"Gehad A.","family":"Basuony","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alshaimaa M. 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The experimental protocol was formally reviewed and approved by the Institutional Review Board (IRB) of the research ethical committee (REC) of the Kasralainy Faculty of Medicine, Cairo University, Giza, Egypt, under approval number N-278-2025. In strict accordance with the institutional review board\u2019s policy governing existing pathology specimens and stored biological investigations collected anonymously, the requirement for active, prospective informed participant consent was deemed unnecessary and officially waived. All laboratory procedures, cultivation protocols, and sample handling workflows were executed in strict compliance with the relevant institutional guidelines, national regulations, and the ethical tenets established by the Declaration of Helsinki.","order":1,"name":"Ethics","label":"Ethics approval and consent to participate","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"None.","order":2,"name":"Ethics","label":"Consent for publication","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"The authors declare no competing interests.","order":3,"name":"Ethics","label":"Competing interests","group":{"name":"EthicsHeading","label":"Declarations"}}],"article-number":"295"}}