{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T14:14:39Z","timestamp":1785334479913,"version":"3.55.0"},"reference-count":35,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T00:00:00Z","timestamp":1785283200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Key Research and Development Program of Shaanxi Province","award":["2024NC-YBXM-180"],"award-info":[{"award-number":["2024NC-YBXM-180"]}]},{"name":"Science and Technology Program of the Yangling Demonstration Zone of Agricultural Hi-Tech Industries","award":["2024SF-01"],"award-info":[{"award-number":["2024SF-01"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Int. J. Mol. Sci.","IJMS"],"abstract":"<jats:p>The seeds of Annona squamosa are widely used in traditional medicine and food, yet their safety profile remains unclear and the principal toxic constituent is unknown. This study aimed to identify the main toxic constituent and elucidate its toxicological mechanisms. A toxicity-guided isolation strategy using zebrafish larvae was employed, combined with morphological, behavioral, biochemical, metabolomic, and transcriptional analyses. Bioassay-directed fractionation identified squamocin, an annonaceous acetogenin, as the principal toxic constituent\u2014the first reported acute toxicant from A. squamosa. Squamocin exerted potent multi-organ toxicity at nanomolar concentrations (LC10 = 62 nM; LC50 = 75 nM), including lethality, developmental teratogenicity, and craniofacial cartilage dysplasia, without substantial growth retardation. Neurotoxicity was dose-dependent: locomotor suppression at 20 nM and motor incoordination at 40 nM. Metabolomics revealed perturbations in amino acid and glycan metabolism, and transcriptional analysis confirmed altered expression of stress-related genes (pipox, myd88, and rigi). These findings establish squamocin as the major toxicant in A. squamosa seeds and provide a mechanistic framework for its broad toxicological spectrum, informing safer use of this material.<\/jats:p>","DOI":"10.3390\/ijms27156802","type":"journal-article","created":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T13:53:31Z","timestamp":1785333211000},"page":"6802","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Squamocin as the Principal Toxic Constituent of Annona squamosa Seeds: Toxicity-Guided Isolation, Developmental and Neurotoxicity, and Metabolic Perturbations"],"prefix":"10.3390","volume":"27","author":[{"given":"Chenyuan","family":"Qi","sequence":"first","affiliation":[{"name":"Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lu","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xue","family":"Li","sequence":"additional","affiliation":[{"name":"Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengling","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Disheng","family":"Wang","sequence":"additional","affiliation":[{"name":"Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7904-2347","authenticated-orcid":false,"given":"Qiang","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Moussa, A.Y., Siddiqui, S.A., Elhawary, E.A., Guo, K., Anwar, S., and Xu, B. (2024). Phytochemical Constituents, Bioactivities, and Applications of Custard Apple (Annona squamosa L.): A Narrative Review. Food Chem., 459.","DOI":"10.1016\/j.foodchem.2024.140363"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Al Kazman, B.S.M., Harnett, J.E., and Hanrahan, J.R. (2022). Traditional Uses, Phytochemistry and Pharmacological Activities of Annonacae. Molecules, 27.","DOI":"10.3390\/molecules27113462"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Yassin, S., Elsohafy, S.M., El-Hawiet, A., Abdel-Kader, M.S., Ghareeb, D.A., Darwish, F.A., and Amer, M.E. (2025). Comparative Phytochemical and Pharmacological Analysis of Two Cultivars of Annona squamosa L. Cultivated in Egypt. npj Sci. Food, 9.","DOI":"10.1038\/s41538-024-00368-6"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1135","DOI":"10.1080\/14786419.2022.2134362","article-title":"Three New Cytotoxic Annonaceous Acetogenins from the Seeds of Annona squamosa","volume":"38","author":"Ma","year":"2024","journal-title":"Nat. Prod. Res."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Alaqeel, N.K., Almalki, W.H., Binothman, N., Aljadani, M., Al-Dhuayan, I.S., Alnamshan, M.M., Almulhim, J., Alqosaibi, A.I., Ajmal, M.R., and Alammari, D.M. (2023). The Inhibitory and Anticancer Properties of Annona squamosa L. Seed Extracts. Braz. J. Biol., 82.","DOI":"10.1590\/1519-6984.268250"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Ferreira, G.G., Quaresma, A.C.S., Brand\u00e3o, D.L.d.N., Marinho, A.M.d.R., Siqueira, J.E.d.S., Correa, K.L., Silva-J\u00fanior, J.O.C., Percario, S., and Dolabela, M.F. (2023). Evaluation of Genotoxicity and Toxicity of Annona muricata L. Seeds and In Silico Studies. Molecules, 28.","DOI":"10.3390\/molecules28010231"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Gonzalez-Lizarraga, F., Boluda, S., Hidalgo, J.R., Avila, C.L., Santos, C.D., Socias, B., Medina, L., Chaumont, H., Akbar, D., and Roze, E. (2025). Protein Coaggregation in Caribbean Atypical Parkinsonism: The Contribution of Annonacin. Neuropathol. Appl. Neurobiol., 51.","DOI":"10.1111\/nan.70026"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Costa, M.F.D., Rosler, T.W., and Hoglinger, G.U. (2024). Exploring the Neuroprotective Potential of Nrf2-pathway Activators against Annonacin Toxicity. Sci. Rep., 14.","DOI":"10.1038\/s41598-024-70837-1"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"4802","DOI":"10.1248\/cpb.36.4802","article-title":"Squamocin, a New Cytotoxic Bis-Tetrahydrofuran Containing Acetogenin from Annona squamosa","volume":"36","author":"Fujimoto","year":"1988","journal-title":"Chem. Pharm. Bull."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Li, X., Jiang, H., Guo, D., Huang, W., Ren, H., and Zhang, Q. (2024). Toxic Features and Metabolomic Intervention of Glabrene, an Impurity Found in the Pharmaceutical Product of Glabridin. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25168985"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Chen, M., Zhao, C., Li, Z., Fan, Q., Lu, S., Tao, X., Lin, Y., Lin, R., and Wu, J. (2023). Investigation of the Applicability of the Zebrafish Model for the Evaluation of Aristolochic Acid-Related Nephrotoxicity. Phytomedicine, 121.","DOI":"10.1016\/j.phymed.2023.155092"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Zhu, R., Liu, C., Wang, J., Zou, L., Yang, F., Chi, X., and Zhu, J. (2023). Nano-TiO2 Aggravates Bioaccumulation and Developmental Neurotoxicity of Difenoconazole in Zebrafish Larvae via Oxidative Stress and Apoptosis: Protective Role of Vitamin C. Ecotoxicol. Environ. Saf., 251.","DOI":"10.1016\/j.ecoenv.2023.114554"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Wang, J., Zhang, Y., Liu, Y., You, Z., Huang, J., Lian, K., Duan, P., and Jiang, Q. (2025). Developmental Toxicity and Transcriptome Analysis of Zebrafish (Danio rerio) Embryos Exposed to Anesthetic Esketamine. Chem. Biol. Interact., 419.","DOI":"10.1016\/j.cbi.2025.111633"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4679","DOI":"10.2147\/IJN.S448860","article-title":"The Remarkable Anti-Breast Cancer Efficacy and Anti-Metastasis by Multifunctional Nanoparticles Co-Loading Squamocin, R848 and IR 780","volume":"19","author":"Wang","year":"2024","journal-title":"Int. J. Nanomed."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Zhu, Y., Liu, Y., Wang, X., Chen, Z., Chen, B., Hu, B., Tang, T., Cheng, H., Liu, X., and Ning, Y. (2025). Squamocin Suppresses Tumor Growth through Triggering an Endoplasmic Reticulum Stress-Associated Degradation of EZH2\/MYC Axis (Adv. Sci. 15\/2025). Adv. Sci., 12.","DOI":"10.1002\/advs.202570109"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Xu, W., Cai, X., Wu, Y., Wen, Y., Su, R., Zhang, Y., Huang, Y., Zheng, Q., Hu, L., and Cui, X. (2023). Biomimetic Single Al-OH Site with High Acetylcholinesterase-like Activity and Self-Defense Ability for Neuroprotection. Nat. Commun., 14.","DOI":"10.1038\/s41467-023-41765-x"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Zhao, W., Gasterich, N., Clarner, T., Voelz, C., Behrens, V., Beyer, C., Fragoulis, A., and Zendedel, A. (2022). Astrocytic Nrf2 Expression Protects Spinal Cord from Oxidative Stress Following Spinal Cord Injury in a Male Mouse Model. J. Neuroinflamm., 19.","DOI":"10.1186\/s12974-022-02491-1"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1017\/S1041610203008676","article-title":"Butyrylcholinesterase: An Important New Target in Alzheimer\u2019s Disease Therapy","volume":"14","author":"Greig","year":"2002","journal-title":"Int. Psychogeriatr."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Chen, X., Guo, W., Lei, L., Guo, Y., Yang, L., Han, J., and Zhou, B. (2021). Bioconcentration and Developmental Neurotoxicity of Novel Brominated Flame Retardants, Hexabromobenzene and Pentabromobenzene in Zebrafish. Environ. Pollut., 268.","DOI":"10.1016\/j.envpol.2020.115895"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Guo, Y., Wu, J., and Guo, W. (2026). The Neuropathological Mechanisms Underlying the Inborn Errors of Lysine Metabolism. Neurobiol. Dis., 220.","DOI":"10.1016\/j.nbd.2026.107315"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Qiao, Y.N., Chen, W.C., Chen, Y.L., Xiang, J., Ke, Y.F., Cao, L., Li, L., Cao, J., Zhao, R., and Zhao, J.Y. (2025). High Maternal Glycine Levels Increase the Risk of Developing Atrial Septal Defect in the Offspring. JACC Basic Transl. Sci., 10.","DOI":"10.1016\/j.jacbts.2025.05.006"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Wang, X., Yue, M., Cheung, J.P.Y., Cheung, P.W.H., Fan, Y., Wu, M., Wang, X., Zhao, S., Khanshour, A.M., and Rios, J.J. (2024). Impaired Glycine Neurotransmission Causes Adolescent Idiopathic Scoliosis. J. Clin. Investig., 134.","DOI":"10.1172\/JCI168783"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Zhao, S., Ling, Y., Zhang, B., Wang, D., and Sun, L. (2025). Integrated Multi-Omics Analysis Reveals the Underlying Molecular Mechanism for the Neurotoxicity of Triclosan in Zebrafish. Ecotoxicol. Environ. Saf., 290.","DOI":"10.1016\/j.ecoenv.2024.117537"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"73393","DOI":"10.1007\/s11356-023-27665-z","article-title":"Endosulfan Affects Embryonic Development Synergistically under Elevated Ambient Temperature","volume":"30","author":"Zaman","year":"2023","journal-title":"Environ. Sci. Pollut. Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"4304","DOI":"10.1021\/acs.est.4c11757","article-title":"Leveraging Zebrafish Embryo Phenotypic Observations to Advance Data-Driven Analyses in Toxicology","volume":"59","author":"Michaelis","year":"2025","journal-title":"Environ. Sci. Technol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Wang, P., Song, Y., Jiang, H., Qi, C., Zhang, X., Wang, D., Li, L., and Zhang, Q. (2025). Sakuranetin, A Laxative Component from Peach Leaves and Its Intervention in Metabolism. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26178112"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Leask, M., Carleton, C., Leeke, B., Newman, T., Antoun, J., Farella, M., and Horsfield, J. (2021). Riboceine Rescues Auranofin-Induced Craniofacial Defects in Zebrafish. Antioxidants, 10.","DOI":"10.3390\/antiox10121964"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"562","DOI":"10.1016\/j.jes.2025.06.044","article-title":"Effect of Amoxicillin on Cartilage and Bone Development in Zebrafish and Their Molecular Mechanisms","volume":"162","author":"Long","year":"2026","journal-title":"J. Environ. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Zhang, Y., Li, X., Liu, Z., Zhao, X., Chen, L., Hao, G., Ye, X., Meng, S., Xiao, G., and Mu, J. (2023). The Neurobehavioral Impacts of Typical Antibiotics toward Zebrafish Larvae. Chemosphere, 340.","DOI":"10.1016\/j.chemosphere.2023.139829"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Issac, P.K., Guru, A., Velayutham, M., Pachaiappan, R., Arasu, M.V., Al-Dhabi, N.A., Choi, K.C., Harikrishnan, R., and Arockiaraj, J. (2021). Oxidative Stress Induced Antioxidant and Neurotoxicity Demonstrated in Vivo Zebrafish Embryo or Larval Model and Their Normalization Due to Morin Showing Therapeutic Implications. Life Sci., 283.","DOI":"10.1016\/j.lfs.2021.119864"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1016\/j.colsurfb.2017.05.078","article-title":"Developmental Neurotoxic Effects of Graphene Oxide Exposure in Zebrafish Larvae (Danio Rerio)","volume":"157","author":"Soares","year":"2017","journal-title":"Colloids Surf. B Biointerfaces"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Zhang, C., Zhang, X., Qiu, H., Song, Q., Wang, Y., Zhang, C., and Zhang, Q. (2025). Fermented Gastrodia elata Bl. Intervenes gut microbiota and amino acid metabolism in zebrafish to promote 5-HT homeostasis against sleep disturbances. Food Res. Int., 217.","DOI":"10.1016\/j.foodres.2025.116757"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"D672","DOI":"10.1093\/nar\/gkae909","article-title":"KEGG: Biological Systems Database as a Model of the Real World","volume":"53","author":"Kanehisa","year":"2025","journal-title":"Nucleic Acids Res."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Picart-Armada, S., Fern\u00e1ndez-Albert, F., Vinaixa, M., Yanes, O., and Perera-Lluna, A. (2018). FELLA: An R Package to Enrich Metabolomics Data. BMC Bioinform., 19.","DOI":"10.1186\/s12859-018-2487-5"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"4859","DOI":"10.1111\/bph.17318","article-title":"Schwann Cell Transient Receptor Potential Ankyrin 1 (TRPA1) Ortholog in Zebrafish Larvae Mediates Chemotherapy-Induced Peripheral Neuropathy","volume":"181","author":"Bellantoni","year":"2024","journal-title":"Br. J. Pharmacol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/15\/6802\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T13:54:08Z","timestamp":1785333248000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/15\/6802"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,29]]},"references-count":35,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["ijms27156802"],"URL":"https:\/\/doi.org\/10.3390\/ijms27156802","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,29]]}}}