Therapeutic effect of baicalein as an antiparasitic agent against Toxoplasma gondii in vitro and in vivo.

The most common medications for the treatment of zoonotic toxoplasmosis are pyrimethamine and sulfadiazine, which may cause serious undesirable side effects. Thus, there is an urgent need to develop novel therapeutics. Baicalein (BAI, C15H10O5) has been shown to perform well against protozoan parasites including Leishmania and Cryptosporidium. In this study, the inhibition efficacy of BAI on Toxoplasma gondii was evaluated using plaque, invasion, and intracellular proliferation assays. BAI effectively inhibited T. gondii (half-maximum inhibitory concentration (IC50)=6.457×10−5 mol/L), with a reduced invasion rate (33.56%) and intracellular proliferation, and exhibited low cytotoxicity (half-maximum toxicity concentration (TC50)=5.929×10−4 mol/L). Further investigation using a mouse model shed light on the inhibitory efficacy of BAI against T. gondii, as well as the potential mechanisms underlying its anti-parasitic effects. The survival time of T. gondii-infected ICR mice treated with BAI was remarkably extended, and their parasite burdens in the liver and spleen were greatly reduced compared with those of the negative control group. Histopathological examination of live sections revealed effective therapeutic outcomes in the treatment groups, with no notable pathological alterations observed. Furthermore, alterations in cytokine levels indicated that BAI not only effectively suppressed the growth of T. gondii but also prevented excessive inflammation in mice. Collectively, these findings underscore the significant inhibitory efficacy of BAI against T. gondii, positioning it as a promising alternative therapeutic agent for toxoplasmosis. 摘要 乙胺嘧啶和磺胺嘧啶是目前治疗人畜共患弓形虫病最常用的药物, 由于二者可能引发严重的不良反应, 因此亟需寻找新的治疗药物。 黄芩素 (baicalein, BAI) 已被证实对原生动物寄生虫 (如利什曼原虫和隐孢子虫) 具有良好的治疗效果。 本研究通过斑块、侵袭和细胞内增殖试验评估 BAI 对刚地弓形虫的抑制效果。 结果显示, BAI 能有效抑制弓形虫的生长, 其半数最大抑制浓度 (IC50) 为 6.457×10−5 mol/L, 显著降低了病原体的侵袭率 (降低 33.56%) 及其在细胞内的增殖能力。 此外, BAI 具有较低的细胞毒性, 其半数最大毒性浓度 (TC50) 为 5.929×10−4 mol/L。 ICR 小鼠模型的实验结果进一步阐明了 BAI 对弓形虫的抑制作用, 并揭示了其在抗寄生虫作用中的潜在机制。 与阴性对照组相比, BAI 处理显著延长了感染弓形虫小鼠的存活时间, 并显著降低了肝脏和脾脏中的寄生虫负荷; 活体切片的组织病理学检查结果显示, BAI 处理组的治疗效果良好, 未观察到明显病理性改变。 此外, 细胞因子水平的变化结果表明, BAI 不仅能有效抑制弓形虫的生长, 还能缓解小鼠过度的炎症反应。 综上所述, 本研究明确了 BAI 在治疗弓形虫感染方面具有显著的抑制作用, 有望作为治疗弓形虫病的潜在替代药剂。 Similar content being viewed by others Data availability statement The authors declare that the experimental data supporting the findings of this study are available within the paper and they have no known competing financial interests or personal relationships that could influence the work presented in this paper.

Ben-Harari RR, Goodwin E, Casoy J, 2017. Adverse event profile of pyrimethamine-based therapy in toxoplasmosis: a systematic review. Drugs R D, 17(4):523–544. https://doi.org/10.1007/s40268-017-0206-8 Biswas A, French T, Düsedau HP, et al., 2017. Behavior of neutrophil granulocytes during Toxoplasma gondii infection in the central nervous system. Front Cell Infect Microbiol, 7:259. https://doi.org/10.3389/fcimb.2017.00259 Chan JKC, 2014. The wonderful colors of the hematoxylineosin stain in diagnostic surgical pathology. Int J Surg Pathol, 22(1):12–32. https://doi.org/10.1177/1066896913517939 Cheraghipour K, Masoori L, Ezzatpour B, et al., 2021. The experimental role of medicinal plants in treatment of Toxoplasma gondii infection: a systematic review. Acta Parasitol, 66(2):303–328. https://doi.org/10.1007/s11686-020-00300-4 Cristaldi C, Saldarriaga Cartagena AM, Ganuza A, et al., 2023. Evaluation of topotecan and 10-hydroxycamptothecin on Toxoplasma gondii: implications on baseline DNA damage and repair efficiency. Int J Parasitol Drugs Drug Resist, 23:120–129. https://doi.org/10.1016/j.ijpddr.2023.11.004 Daher D, Shaghlil A, Sobh E, et al., 2021. Comprehensive overview of Toxoplasma gondii-induced and associated diseases. Pathogens, 10(11):1351. https://doi.org/10.3390/pathogens10111351 Dai CS, Li H, Wang Y, et al., 2021. Inhibition of oxidative stress and ALOX12 and NF-κB pathways contribute to the protective effect of baicalein on carbon tetrachloride-induced acute liver injury. Antioxidants (Basel), 10(6):976. https://doi.org/10.3390/antiox10060976 Daniel V, Naujokat C, Sadeghi M, et al., 2008. Observational support for an immunoregulatory role of CD3+CD4+CD25+IFN-γ+ blood lymphocytes in kidney transplant recipients with good long-term graft outcome. Transpl Int, 21(7):646–660. https://doi.org/10.1111/j.1432-2277.2008.00662.x Das BB, Sen N, Roy A, et al., 2006. Differential induction of Leishmania donovani bi-subunit topoisomerase I–DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I. Nucleic Acids Res, 34(4):1121–1132. https://doi.org/10.1093/nar/gkj502 de Oliveira MR, Nabavi SF, Habtemariam S, et al., 2015. The effects of baicalein and baicalin on mitochondrial function and dynamics: a review. Pharmacol Res, 100:296–308. https://doi.org/10.1016/j.phrs.2015.08.021 de Oliveira TC, Oliveira Silva DA, Rostkowska C, et al., 2009. Toxoplasma gondii: effects of Artemisia annua L. on susceptibility to infection in experimental models in vitro and in vivo. Exp Parasitol, 122(3):233–241. https://doi.org/10.1016/j.exppara.2009.04.010 Dimier-Poisson I, Aline F, Mévélec MN, et al., 2003. Protective mucosal Th2 immune response against Toxoplasma gondii by murine mesenteric lymph node dendritic cells. Infect Immun, 71(9):5254–5265. https://doi.org/10.1128/iai.71.9.5254-5265.2003 Dinda B, Dinda S, Dassharma S, et al., 2017. Therapeutic potentials of baicalin and its aglycone, baicalein against inflammatory disorders. Eur J Med Chem, 131:68–80. https://doi.org/10.1016/j.ejmech.2017.03.004 Djurković-Djaković O, Milenković V, Nikolić A, et al., 2002. Efficacy of atovaquone combined with clindamycin against murine infection with a cystogenic (Me49) strain of Toxoplasma gondii. J Antimicrob Chemother, 50(6):981–987. https://doi.org/10.1093/jac/dkf251 Du KG, Lu F, Xie CZ, et al., 2022. Toxoplasma gondii infection induces cell apoptosis via multiple pathways revealed by transcriptome analysis. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 23(4):315–327. https://doi.org/10.1631/jzus.B2100877 Egan CE, Sukhumavasi W, Butcher BA, et al., 2009. Functional aspects of Toll-like receptor/MyD88 signalling during protozoan infection: focus on Toxoplasma gondii. Clin Exp Immunol, 156(1):17–24. https://doi.org/10.1111/j.1365-2249.2009.03876.x Fichera ME, Bhopale MK, Roos DS, 1995. In vitro assays elucidate peculiar kinetics of clindamycin action against Toxoplasma gondii. Antimicrob Agents Chemother, 39(7):1530–1537. https://doi.org/10.1128/aac.39.7.1530 Fox BA, Bzik DJ, 2002. De novo pyrimidine biosynthesis is required for virulence of Toxoplasma gondii. Nature, 415(6874):926–929. https://doi.org/10.1038/415926a Gay G, Braun L, Brenier-Pinchart MP, et al., 2016. Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-γ-mediated host defenses. J Exp Med, 213(9):1779–1798. https://doi.org/10.1084/jem.20160340 Hall AO, Beiting DP, Tato C, et al., 2012. The cytokines interleukin 27 and interferon-γ promote distinct Treg cell populations required to limit infection-induced pathology. Immunitty, 37(3):511–523. https://doi.org/10.1016/j.immuni.2012.06.014 Haverkos HW, 1987. Assessment of therapy for toxoplasma encephalitis the TE study group. Am J Med, 82(5):907–914. https://doi.org/10.1016/0002-9343(87)90151-3 He XL, Wang YH, Gao M, et al., 2009. Baicalein protects rat brain mitochondria against chronic cerebral hypoperfusion-induced oxidative damage. Brain Res, 1249:212–221. https://doi.org/10.1016/j.brainres.2008.10.005 He XX, Wei ZK, Zhou ES, et al., 2015. Baicalein attenuates inflammatory responses by suppressing TLR4 mediated NF-κB and MAPK signaling pathways in LPS-induced mastitis in mice. Int Immunopharmacol, 28(1):470–476. https://doi.org/10.1016/j.intimp.2015.07.012 Hopper AT, Brockman A, Wise A, et al., 2019. Discovery of selective Toxoplasma gondii dihydrofolate reductase inhibitors for the treatment of toxoplasmosis. J Med Chem, 62(3):1562–1576. https://doi.org/10.1021/acs.jmedchem.8b01754 Hou JZ, Schindler U, Henzel WJ, et al., 1994. An interleukin-4-induced transcription factor: IL-4 Stat. Science, 265(5179):1701–1706. https://doi.org/10.1126/science.8085155 Hu QC, Zhang WW, Wu Z, et al., 2021. Baicalin and the liver-gut system: pharmacological bases explaining its therapeutic effects. Pharmacol Res, 165:105444. https://doi.org/10.1016/j.phrs.2021.105444 Jiang LP, Liu B, Hou SJ, et al., 2022. Discovery and evaluation of chalcone derivatives as novel potential anti-Toxoplasma gondii agents. Eur J Med Chem, 234:114244. https://doi.org/10.1016/j.ejmech.2022.114244 Jin Z, Ma JB, Zhu G, et al., 2019. Discovery of novel anticryptosporidial activities from natural products by in vitro high-throughput phenotypic screening. Front Microbiol, 10:1999. https://doi.org/10.3389/fmicb.2019.01999 Johari J, Kianmehr A, Mustafa MR, et al., 2012. Antiviral activity of baicalein and quercetin against the Japanese encephalitis virus. Int J Mol Sci, 13(12):16785–16795. https://doi.org/10.3390/ijms131216785 Kovacs JA, 1992. Efficacy of atovaquone in treatment of toxoplasmosis in patients with AIDS. Lancet, 340(8820):637–638. https://doi.org/10.1016/0140-6736(92)92172-c Lagal V, Dinis M, Cannella D, et al., 2015. AMA1-deficient Toxoplasma gondii parasites transiently colonize mice and trigger an innate immune response that leads to long-lasting protective immunity. Infect Immun, 83(6):2475–2486. https://doi.org/10.1128/iai.02606-14 le Gros G, Ben-Sasson SZ, Seder R, et al., 1990. Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4-producing cells. J Exp Med, 172(3):921–929. https://doi.org/10.1084/jem.172.3.921 Lee IK, Kang KA, Zhang R, et al., 2011. Mitochondria protection of baicalein against oxidative damage via induction of manganese superoxide dismutase. Environ Toxicol Pharmacol, 31(1):233–241. https://doi.org/10.1016/j.etap.2010.11.002 Li W, Grech J, Stortz JF, et al., 2022. A splitCas9 phenotypic screen in Toxoplasma gondii identifies proteins involved in host cell egress and invasion. Nat Microbiol, 7(6):882–895. https://doi.org/10.1038/s41564-022-01114-y Li-Weber M, 2009. New therapeutic aspects of flavones: the anticancer properties of Scutellaria and its main active constituents Wogonin, Baicalein and Baicalin. Cancer Treat Rev, 35(1):57–68. https://doi.org/10.1016/j.ctrv.2008.09.005 Liang W, Huang XB, Chen WQ, 2017. The effects of baicalin and baicalein on cerebral ischemia: a review. Aging Dis, 8(6):850–867. https://doi.org/10.14336/ad.2017.0829 Liu G, Li ZF, Wei Y, et al., 2014. Direct detection of FoxP3 expression in thymic double-negative CD4−CD8− cells by flow cytometry. Sci Rep, 4:5781. https://doi.org/10.1038/srep05781 Liu SX, Wu MM, Hua QQ, et al., 2020. Two old drugs, NVP-AEW541 and GSK-J4, repurposed against the Toxoplasma gondii RH strain. Parasit Vectors, 13:242. https://doi.org/10.1186/s13071-020-04094-2 Lu CX, Liu XY, Liu J, et al., 2022. Immunocompetent rabbits infected with Cryptosporidium cuniculus as an animal model for anti-cryptosporidial drug testing. Int J Parasitol, 52(4):205–210. https://doi.org/10.1016/j.ijpara.2021.10.006 Luo Q, Ye JQ, Zeng LL, et al., 2017. Elevated expression of TIGIT on CD3+CD4+ T cells correlates with disease activity in systemic lupus erythematosus. Allergy Asthma Clin Immunol, 13:15. https://doi.org/10.1186/s13223-017-0188-7 Luo Z, Kuang XP, Zhou QQ, et al., 2020. Inhibitory effects of baicalein against herpes simplex virus type 1. Acta Pharm Sin B, 10(12):2323–2338. https://doi.org/10.1016/j.apsb.2020.06.008 Melchor SJ, Ewald SE, 2019. Disease tolerance in Toxoplasma infection. Front Cell Infect Microbiol, 9:185. https://doi.org/10.3389/fcimb.2019.00185 Mitchell RE, Hassan M, Burton BR, et al., 2017. IL-4 enhances IL-10 production in Th1 cells: implications for Th1 and Th2 regulation. Sci Rep, 7:11315. https://doi.org/10.1038/s41598-017-11803-y Montazeri M, Sharif M, Sarvi S, et al., 2017. A systematic review of in vitro and in vivo activities of anti-Toxoplasma drugs and compounds (2006–2016). Front Microbiol, 8:25. https://doi.org/10.3389/fmicb.2017.00025 Munoz M, Liesenfeld O, Heimesaat MM, 2011. Immunology of Toxoplasma gondii. Immunol Rev, 240(1):269–285. https://doi.org/10.1111/j.1600-065X.2010.00992.x Muscate F, Woestemeier A, Gagliani N, 2021. Functional heterogeneity of CD4+ T cells in liver inflammation. Semin Immunopathol, 43(4):549–561. https://doi.org/10.1007/s00281-021-00881-w Nedergaard BS, Ladekarl M, Thomsen HF, et al., 2007. Low density of CD3+, CD4+ and CD8+ cells is associated with increased risk of relapse in squamous cell cervical cancer. Br J Cancer, 97(8):1135–1138. https://doi.org/10.1038/sj.bjc.6604001 Neyer LE, Grunig G, Fort M, et al., 1997. Role of interleukin-10 in regulation of T-cell-dependent and T-cell-independent mechanisms of resistance to Toxoplasma gondii. Infect Immun, 65(5):1675–1682. https://doi.org/10.1128/iai.65.5.1675-1682.1997 Olias P, Etheridge RD, Zhang Y, et al., 2016. Toxoplasma effector recruits the Mi-2/NuRD complex to repress STAT1 transcription and block IFN-γ-dependent gene expression. Cell Host Microbe, 20(1):72–82. https://doi.org/10.1016/j.chom.2016.06.006 Papanastasatou M, Verykokakis M, 2023. Innate-like T lymphocytes in chronic liver disease. Front Immunol, 14:1114605. https://doi.org/10.3389/fimmu.2023.1114605 Park W, Jang H, Kim HS, et al., 2024. Therapeutic efficacy and anti-inflammatory mechanism of baicalein on endometriosis progression in patient-derived cell line and mouse model. Phytomedicine, 130:155469. https://doi.org/10.1016/j.phymed.2024.155469 Patwardhan RS, Sharma D, Thoh M, et al., 2016. Baicalein exhibits anti-inflammatory effects via inhibition of NF-κB transactivation. Biochem Pharmacol, 108:75–89. https://doi.org/10.1016/j.bcp.2016.03.013 Piketty C, Derouin F, Rouveix B, et al., 1990. In vivo assessment of antimicrobial agents against Toxoplasma gondii by quantification of parasites in the blood, lungs, and brain of infected mice. Antimicrob Agents Chemother, 34(8):1467–1472. https://doi.org/10.1128/aac.34.8.1467 Platanias LC, 2005. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol, 5(5):375–386. https://doi.org/10.1038/nri1604 Porter SB, Sande MA, 1992. Toxoplasmosis of the central nervous system in the acquired immunodeficiency syndrome. N Engl J Med, 327(23):1643–1648. https://doi.org/10.1056/nejm199212033272306 Qi ZL, Yin F, Lu LN, et al., 2013. Baicalein reduces lipopolysaccharide-induced inflammation via suppressing JAK/STATs activation and ROS production. Inflamm Res, 62(9):845–855. https://doi.org/10.1007/s00011-013-0639-7 Rathore AS, Kumar S, Konwar R, et al., 2014. CD3+, CD4+ & CD8+ tumour infiltrating lymphocytes (TILs) are predictors of favourable survival outcome in infiltrating ductal carcinoma of breast. Indian J Med Res, 140(3):361–369. Reslan A, Haddad JG, Moukendza Koundi L, et al., 2021. Zika virus growth in human kidney cells is restricted by an elevated glucose level. Int J Mol Sci, 22(5):2495. https://doi.org/10.3390/ijms22052495 Romand S, Pudney M, Derouin F, 1993. In vitro and in vivo activities of the hydroxynaphthoquinone atovaquone alone or combined with pyrimethamine, sulfadiazine, clarithromycin, or minocycline against Toxoplasma gondii. Antimicrob Agents Chemother, 37(11):2371–2378. https://doi.org/10.1128/aac.37.11.2371 Saito H, Murata Y, Nonaka M, et al., 2020. Screening of a library of traditional Chinese medicines to identify compounds and extracts which inhibit Toxoplasma gondii growth. J Vet Med Sci, 82(2):184–187. https://doi.org/10.1292/jvms.19-0241 Soheilian M, Ramezani A, Azimzadeh A, et al., 2011. Randomized trial of intravitreal clindamycin and dexamethasone versus pyrimethamine, sulfadiazine, and prednisolone in treatment of ocular toxoplasmosis. Ophthalmology, 118(1):134–141. https://doi.org/10.1016/j.ophtha.2010.04.020 Sowndhararajan K, Deepa P, Kim M, et al., 2017. Baicalein as a potent neuroprotective agent: a review. Biomed Pharmacother, 95:1021–1032. https://doi.org/10.1016/j.biopha.2017.08.135 Swierczynski B, Bessieres MH, Cassaing S, et al., 2000. Inhibitory activity of anti-interleukin-4 and anti-interleukin-10 antibodies on Toxoplasma gondii proliferation in mouse peritoneal macrophages cocultured with splenocytes from infected mice. Parasitol Res, 86(2):151–157. https://doi.org/10.1007/s004360050024 Tomura M, Yu WG, Ahn HJ, et al., 1999. A novel function of Vα14+ CD4+ NKT cells: stimulation of IL-12 production by antigen-presenting cells in the innate immune system. J Immunol, 163(1):93–101. https://doi.org/10.4049/jimmunol.163.1.93 Trinchieri G, 1997. Cytokines acting on or secreted by macrophages during intracellular infection (IL-10, IL-12, IFN-γ). Curr Opin Immunol, 9(1):17–23. https://doi.org/10.1016/s0952-7915(97)80154-9 Wang X, Xie L, Long JY, et al., 2022. Therapeutic effect of baicalin on inflammatory bowel disease: a review. J Ethnopharmacol, 283:114749. https://doi.org/10.1016/j.jep.2021.114749 Wang YJ, Han E, Xing QH, et al., 2015. Baicalein upregulates DDIT4 expression which mediates mTOR inhibition and growth inhibition in cancer cells. Cancer Lett, 358(2):170–179. https://doi.org/10.1016/j.canlet.2014.12.033 Xin LY, Gao JL, Lin HC, et al., 2020. Regulatory mechanisms of baicalin in cardiovascular diseases: a review. Front Pharmacol, 11:583200. https://doi.org/10.3389/fphar.2020.583200 Yang XY, Huang B, Chen JP, et al., 2012. In vitro effects of aqueous extracts of Astragalus membranaceus and Scutellaria baicalensis GEORGI on Toxoplasma gondii. Parasitol Res, 110(6):2221–2227. https://doi.org/10.1007/s00436-011-2752-2 Yarovinsky F, 2008. Toll-like receptors and their role in host resistance to Toxoplasma gondii. Immunol Lett, 119(1–2):17–21. https://doi.org/10.1016/j.imlet.2008.05.007 Yun MY, Yang JH, Kim DK, et al., 2010. Therapeutic effects of baicalein on atopic dermatitis-like skin lesions of NC/Nga mice induced by dermatophagoides pteronyssinus. Int Immunopharmacol, 10(9):1142–1148. https://doi.org/10.1016/j.intimp.2010.06.020 Zeng Y, Song C, Ding X, et al., 2007. Baicalin reduces the severity of experimental autoimmune encephalomyelitis. Braz J Med Biol Res, 40(7):1003–1010. https://doi.org/10.1590/s0100-879x2006005000115 Zhang JL, Si HF, Li B, et al., 2019. Myrislignan exhibits activities against Toxoplasma gondii RH strain by triggering mitochondrial dysfunction. Front Microbiol, 10:2152. https://doi.org/10.3389/fmicb.2019.02152 Zhao XY, Kong DW, Zhou QM, et al., 2021. Baicalein alleviates depression-like behavior in rotenone-induced Parkinson’s disease model in mice through activating the BDNF/TrkB/CREB pathway. Biomed Pharmacother, 140:111556. https://doi.org/10.1016/j.biopha.2021.111556 Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 32370997), the Key Projects Jointly Constructed by the Ministry and the Province of Zhejiang Medical and Health Science and Technology Project (No. WKJ-ZJ-2545), the Chinese Medicine Science and Technology Program of Zhejiang Province (No. 2024ZL367), the Health Commission of Zhejiang Province (No. 2024KY923), and the Foundation of GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals (No. GTKF(23)001), China. Author information Authors and Affiliations Contributions Songrui WU and Yingmei LAI collaborated on both the in vitro and in vivo experiments. Zhong’ao ZHANG and Jianzu DING made significant contributions to the in vivo experiments. Songrui WU and Haojie DING played a substantial role in drafting the manuscript. Shaohong LU, Huayue YE, and Xunhui ZHUO were significantly involved in designing the experiments and revising the manuscript. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. Corresponding authors Ethics declarations Songrui WU, Yingmei LAI, Zhong’ao ZHANG, Jianzu DING, Shaohong LU, Huayue YE, Haojie DING, and Xunhui ZHUO declare that they have no conflicts of interest. Mice were fed in accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals of the Chinese Ministry of Science and Technology. The experimental procedures were approved by the Animal Experimentation Ethics Committee of Hangzhou Medical College (No. 2021-134). Additional information Supplementary information Fig. S1 Electronic supplementary material Rights and permissions About this article Cite this article Wu, S., Lai, Y., Zhang, Z. et al. Therapeutic effect of baicalein as an antiparasitic agent against Toxoplasma gondii in vitro and in vivo. J. Zhejiang Univ. Sci. B 26, 1086–1102 (2025). https://doi.org/10.1631/jzus.B2400235 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1631/jzus.B2400235