Systematic evaluation of the anti-Toxoplasma gondii activity of benzoyl metronidazole in vitro and in vivo

doi:10.12140/j.issn.1000-7423.2026.03.015

Abstract

Abstract:

Objective To systematically evaluate the anti-Toxoplasma gondii activity and safety of benzoyl metronidazole (BMZ). Methods Vero cells were treated with BMZ at different concentrations (50, 100, 200, 400, 500, 600, 800, 1 000 μg/mL), and the cytotoxicity of BMZ to Vero cells was detected by cell counting kit-8 (CCK-8) assay. After inoculation with Toxoplasma tachyzoites, Vero cells were treated with BMZ at the above concentrations, and the killing effect of BMZ on Toxoplasma was determined by CCK-8 assay. Vero cells inoculated with Toxoplasma tachyzoites were treated with 50 μg/mL BMZ, and the quantitative and morphological changes of Toxoplasma tachyzoites were observed via Giemsa staining and scanning electron microscopy. Kunming mice were randomly divided into low-dose BMZ group (LD-BMZ group), medium-dose BMZ group (MD-BMZ group), high-dose BMZ group (HD-BMZ group), sulfadiazine sodium group (SD-Na group) and negative control group (NC group). All mice were intraperitoneally inoculated with 1 × 10⁴ Toxoplasma tachyzoites per mL. Four hours after infection, mice were intragastrically administered continuously for 5 days: the LD-BMZ, MD-BMZ and HD-BMZ groups received BMZ at final doses of 12.5, 25 and 50 mg/kg, respectively, the SD-Na group received SD-Na at a final dose of 50 mg/kg, and the NC group received an equal volume of corn oil. On day 7 post-infection, the survival rates of mice were recorded, and the parasite load in mice ascites was counted using a hemocytometer. The heart, liver, spleen, lung, kidney and brain tissues of mice in the MD-BMZ group and NC group were collected. After DNA extraction, qPCR was performed to detect the parasite load in each tissue, and the tissues were processed into pathological sections for HE staining to observe the histopathological changes. GraphPad Prism 9 software was used for data analysis and plotting, and independent sample t-test or one-way ANOVA was adopted for significance analysis. Results The cytotoxicity assay showed that the CC₅₀ of BMZ to Vero cells was 155.67 μg/mL; the cell viability was (108.78 ± 1.00)% when BMZ concentration was 50 μg/mL, and decreased to (81.22 ± 3.34)% at 100 μg/mL. Therefore, 50 μg/mL was determined as the maximum safe concentration of BMZ. The in vitro anti-parasitic assay showed that BMZ could inhibit the proliferation of intracellular Toxoplasma tachyzoites, and the inhibition rate increased with the elevation of BMZ concentration; the inhibition rate of BMZ against Toxoplasma reached 14.3% at 50 μg/mL. Giemsa staining results showed that the number of Toxoplasma tachyzoites in cells of the BMZ group was reduced. Scanning electron microscopy observation showed that normal Toxoplasma tachyzoites presented typical spindle or crescent structures with complete contours, plump morphology and smooth surface; while after BMZ treatment, the structural integrity of Toxoplasma was completely lost, with blurred contours and destroyed cell membrane. The in vivo anti-parasitic assay showed that on day 7 post Toxoplasma infection, the survival rates of mice in the LD-BMZ, MD-BMZ, HD-BMZ, SD-Na and NC groups were 2/10, 3/10, 2/10, 2/10 and 1/10, respectively, with no statistically significant difference (χ² = 3.19, P > 0.05); the ascites parasite loads were (15.00 ± 5.00) × 10⁴, (6.67 ± 2.89) × 10⁴, (6.67 ± 2.89) × 10⁴, (8.33 ± 2.89) × 10⁴ and (1 766.67 ± 2.33) × 10⁴ parasites per mL respectively, with statistically significant difference [F(4,10) = 123 320, P < 0.01]. The qPCR results showed that on day 7 post infection, the relative Toxoplasma parasite loads in the heart, liver, spleen, lung, kidney and brain of mice in the MD-BMZ group were 2.32 ± 0.85, 2.32 ± 0.76, 1.00 ± 0.45, 1.00 ± 0.52, 4.46 ± 0.98 and 1.59 ± 0.63 respectively, all significantly lower than 7.28 ± 1.25, 7.11 ± 1.18, 3.46 ± 0.95, 7.28 ± 1.22, 9.37 ± 1.36 and 8.67 ± 1.29 in the NC group (t = 6.56, 6.83, 4.68, 9.47, 5.86, 9.86, all P < 0.01). HE staining showed that BMZ treatment significantly reduced the inflammatory cell infiltration and granulomatous lesions in the spleen, kidney and liver, without causing obvious drug-related damage. Conclusion BMZ exhibited excellent anti-Toxoplasma activity both in vivo and in vitro, with superior efficacy to SD-Na and high safety at the medium dose.

Key words: Toxoplasma gondii, Benzoyl metronidazole, Anti-parasitic activity, Mouse model, Histopathology

CLC Number:

R531.8

BAO Bibo, ZHAI Bintao, SUN Wankui, CHEN Guohui, LI Qinzhao, ZHANG Jiyu, CHEN Shiming, SUN Xiaolin, WANG Zexiang. Systematic evaluation of the anti-Toxoplasma gondii activity of benzoyl metronidazole in vitro and in vivo[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2026, 44(3): 413-420.

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References 22

[1]	Ko?odziej-Sobocińska M, Holec-G?sior L, Ferra B, et al. A long-term serological survey of Toxoplasma gondii in European Bison (Bison bonasus)-the largest European herbivore[J]. Vet Res Commun, 2025, 50(1): 66.
[2]	Paraoan CEM, Santos CAG, Obusan MCM. Molecular detection of Toxoplasma gondii in irrigation waters and agricultural soils of Central Luzon, Philippines: implications to human health[J]. J Environ Sci Heal Part B, 2025, 60(10): 462-472.
[3]	Pielok ?, Sa?amatin R, Swarcewicz J, et al. Toxocarosis, toxoplasmosis and ocular dirofilariasis diagnosed in a Polish farmer from Wielkopolska Voivodeship-a case description[J]. BMC Ophthalmol, 2025, 25(1): 639.
[4]	Allam AF, Shehab AY, Ahmed NA, et al. IgG avidity and placenta real-time PCR in detection of active maternal toxoplasmosis: relation to pregnancy outcomes[J]. Exp Parasitol, 2025, 279: 109065.
[5]	Moafa HN, Mobarki A, Moafa S, et al. Assessment of awareness, knowledge, and self-reported prevalence of toxoplasmosis in the tropical zone of Saudi Arabia: a cross-sectional study[J]. Trop Med Infect Dis, 2025, 10(11): 323.
[6]	Kwain S, Awasthi V, Islam R, et al. Oxadiargyl analogs as potent inhibitors of Toxoplasma gondii protoporphyrinogen oxidase[J]. RSC Med Chem, 2026, 17(1): 343-353.
[7]	da Silva Siqueira V, Santos Anjo Reis T, Rodrigues Rezende Ferreira S, et al. Evaluation of ethanolic extract and isolated α-bisabolol from Siparuna guianensis in the treatment of experimental neurotoxoplasmosis[J]. Exp Parasitol, 2025, 278: 109045.
[8]	dos Santos-Destro YM, Nunes AP, da Silva Sanfelice RA, et al. Caffeic acid reduces Toxoplasma gondii proliferation in human extravillous trophoblast cells (HTR8/SVneo) through induction of pro-inflammatory cytokines and a death process suggestive of apoptosis[J]. Microbes Infect, 2025, 27(8): 105584.
[9]	胡忆诺, 汪梦蝶, 施春阳, 等. 气相色谱法同时测定苯酰甲硝唑原料药中乙醇甲苯和吡啶残留量[J]. 医药导报, 2022(1): 108-111.
	Hu YN, Wang MD, Shi CY, et al. Simultaneous determination of ethanol, toluene, and pyridine residues in benzoyl metronidazole by gas chromatography[J]. Her Med, 2022(1): 108-111. (in Chinese)
[10]	翟斌涛, 马元元, 谢世臣, 等. 莫能菌素钠、磺胺嘧啶和蒿甲醚体内外抗弓形虫RH株速殖子的效果分析[J]. 中国兽医科学, 2020, 50(5): 624-630.
	Zhai BT, Ma YY, Xie SC, et al. Analysis of in vivo and in vitro effects of monensin natrium, sulfadiazine and artemether on anti-Toxoplasma gondii tachyzoite of RH strain[J]. Chin Vet Sci, 2020, 50(5): 624-630. (in Chinese)
[11]	陈梁元, 莫娇, 曾庆源, 等. 抗弓形虫药物研究进展[J]. 中国人兽共患病学报, 2023, 39(2): 161-171.
	Chen LY, Mo J, Zeng QY, et al. Research progress in anti-Toxoplasma gondii drugs[J]. Chin J Zoonoses, 2023, 39(2): 161-171. (in Chinese)
[12]	Nie LB, Cong W, He JJ, et al. Global proteomic profiling of multiple organs of cats (Felis catus) and proteome-transcriptome correlation during acute Toxoplasma gondii infection[J]. Infect Dis Poverty, 2022, 11(1): 96.
[13]	张吉丽, 司鸿飞, 李冰, 等. 抗弓形虫药物应用现状与研究进展[J]. 中国兽医科学, 2019, 49(8): 1054-1060.
	Zhang JL, Si HF, Li B, et al. Current situation and research progress of anti-Toxoplasma drugs[J]. Chin Vet Sci, 2019, 49(8): 1054-1060. (in Chinese)
[14]	张吉丽. 氯硝柳胺与肉豆蔻木脂素抗弓形虫活性及机理[D]. 北京: 中国农业科学院, 2020: 1-103.
	Zhang JL. The activity and mechanisms of niclosamide and myrislignan against Toxoplasma gondii[D]. Beijing: Chinese Academy of Agricultural Sciences, 2020: 1-103. (in Chinese)
[15]	Lamp KC, Freeman CD, Klutman NE, et al. Pharmacokinetics and pharmacodynamics of the nitroimidazole antimicrobials[J]. Clin Pharmacokinet, 1999, 36(5): 353-373.
[16]	Shahzad I, Alasmari MS, Zamir A, et al. Clinical pharmacokinetics of metronidazole: a systematic review and meta-analysis[J]. Antimicrob Agents Chemother, 2025, 69(9): e0190424.
[17]	Chew WK, Segarra I, Ambu S, et al. Significant reduction of brain cysts caused by Toxoplasma gondii after treatment with spiramycin coadministered with metronidazole in a mouse model of chronic toxoplasmosis[J]. Antimicrob Agents Chemother, 2012, 56(4): 1762-1768.
[18]	王芳芳, 王梅. 红藤汤加减灌肠联合苯酰甲硝唑对腹部手术患者术后盆腔黏连发生率、血液流变学与炎症指标的影响[J]. 长春中医药大学学报, 2021, 37(5): 1047-1050.
	Wang FF, Wang M. Effect of the Enema therapy with the modified Hongteng Decoctions combined with benzoyl metronidazole on the incidence of postoperative pelvic adhesions, hemorheology and inflammation indexes in patients undergoing an abdominal surgery[J]. J Changchun Univ Chin Med, 2021, 37(5): 1047-1050. (in Chinese)
[19]	赵瑞娟, 陈永波, 刘华. 苯酰甲硝唑对幽门螺杆菌感染胃溃疡患者血清多肽激素及胃泌素胃蛋白酶的影响[J]. 山西医药杂志, 2018, 47(3): 315-318.
	Zhao RJ, Chen YB, Liu H. Effects of benzoyl metronidazole on serum peptide hormones, gastrin and pepsin in patients with gastric ulcer and Helicobacter pylori infection[J]. Shanxi Med J, 2018, 47(3): 315-318. (in Chinese)
[20]	赵清平, 易宏英. 苯酰甲硝唑干混悬剂治疗细菌性阴道病临床观察[J]. 中国临床医生杂志, 2015, 43(6): 65-67.
	Zhao QP, Yi HY. Clinical observation of benzoyl metronidazole dry suspension in the treatment of bacterial vaginosis[J]. Chin J Clin, 2015, 43(6): 65-67. (in Chinese)
[21]	Houghton GW, Hundt HK, Muller FO, et al. A comparison of the pharmacokinetics of metronidazole in man after oral administration of single doses of benzoylmetronidazole and metronidazole[J]. Br J Clin Pharmacol, 1982, 14(2): 201-206.
[22]	Ogunbanwo JA, Agbonlahor DE, Adamu A, et al. Effects of anti-protozoal drugs and histopathological studies on trypanosome species[J]. FEMS Immunol Med Microbiol, 2001, 30(1): 73-83.