Acta Parasitologica

, Volume 64, Issue 4, pp 873–886 | Cite as

Optimizing and Evaluating the Antihelminthic Activity of the Biocompatible Zinc Oxide Nanoparticles Against the Ascaridid Nematode, Parascaris equorum In Vitro

  • Kareem MorsyEmail author
  • Sohair Fahmy
  • Ayman Mohamed
  • Sara Ali
  • Manal El–Garhy
  • Mohammed Shazly
Original Paper



In the present study, the effect of different biocompatible concentrations from ZnO nanoparticles (ZnO NPs) on the physiological state and surface topography of the nematode P. equorum was determined in vitro.


Different concentrations of ZnO NPs (100, 200, 300 and 400 mg/l) synthesized using the egg white were prepared followed by the incubation of parasitic worms with these concentrations in vitro. The physiological state of treated worms such as oxidative stress markers, enzymatic activities and biochemical parameters in addition to the surface topography was determined and compared with control untreated worms.


In comparison to control worms, it was observed that at high concentrations of ZnO NPs, most of the treated worms showed an increase in the levels of ALT, AST and ALP (worm muscle damage, and gonad injury); enhancement of the total protein content (worm cellular dysfunction); significant increase in MDA level (free radical-mediated worm cell membrane damage); depletion in GST and GSH activities (reduced ability to clear toxic compounds like lipid peroxides); CAT depletion (superoxide dismutase and hydrogen peroxide toxicity) and NO increase (detoxification activity and stressful conditions on worms). SEM showed that there was a modified morphological appearance in the surface of treated worms; lips were wrinkled with irregularly arranged denticles, weathering of cuticle, bursts of cuticle layers, disruption of surface annulations and erosion of surface papillae of male around the cloacal opening.


ZnO NPs at environmentally relevant concentrations achieved a significant antihelminthic activity against P. equorum which represents a successful model used in parasite control experiments.


Antihelminthic effect ZnO NPs Biocompatibility Nematoda Parascaris equorum Physiological state 



The authors extend their appreciation to the Deanship of the Scientific Research at King Khalid University for funding this work through Research group Project under Grant number (R.G.P.1–56–39).

Compliance with Ethical Standards

Conflict of Interests

No conflict of interests regarding the publication of this article is declared.

Animal Rights Statement

The experiments on animals were conducted in accordance with the local Ethical Committee laws and regulations with regard to care and use of laboratory animals.


  1. 1.
    Agarwal H, Kumar V, Shanmugam R (2017) A review on green synthesis of zinc oxide nanoparticles: an eco-friendly approach. Res Eff Technol 3(4):406–413. CrossRefGoogle Scholar
  2. 2.
    Ali D, Alarifi S, Kumar S, Ahamed M, Siddiqui MA (2012) Oxidative stress and genotoxic effect of zinc oxide nanoparticles in freshwater snail Lymnaea luteola L. Aquat Toxicol 124–125:83–90. CrossRefPubMedGoogle Scholar
  3. 3.
    Alves MM, Andrade SM, Grenho L, Fernandes MH, Santos C, Montemor MF (2019) Influence of apple phytochemicals in ZnO nanoparticles formation, photoluminescence and biocompatibility for biomedical applications. Mater Sci Eng C Mater Biol Appl 101:76–87. CrossRefPubMedGoogle Scholar
  4. 4.
    Amni F, Farahnak A, Golmohammadi T, Eshraghian MR, Rad MBM (2015) The effect of Triclabendazole on ALT enzyme activity in Fasciola hepatica helminths and parasitized sheep liver tissues. J Med Microbiol Infec Dis 3(1–2):1–5Google Scholar
  5. 5.
    Barrett J, Brophy PM (2000) Ascaris haemoglobin: new tricks for an old protein. Parasitol Today 16(3):90–91CrossRefGoogle Scholar
  6. 6.
    Bascal ZA, Cunningham JM, Holden-Dye L, O’Shea M, Walker RJ (2001) Characterization of a putative nitric oxide synthase in the neuromuscular system of the parasitic nematode, Ascaris suum. Parasitology 122:219–231CrossRefGoogle Scholar
  7. 7.
    Becker SL, Liwanag HJ, Snyder JS, Akogun O, Belizario V Jr, Freeman MC, Gyorkos TW, Imtiaz R, Keiser J, Krolewiecki A, Levecke B, Mwandawiro C, Pullan RL, Addiss DG, Utzinger J (2018) Toward the 2020 goal of soil-transmitted helminthiasis control and elimination. PLoS Negl Trop Dis 12(8):e0006606. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Belfield A, Goldberg DM (1971) Colourimetric determination of alkaline phosphatase activity. Enzyme 12:561–568CrossRefGoogle Scholar
  9. 9.
    Behnke JM, Buttle DJ, Stepek G, Lowe A, Duce IR (2008) Developing novel anthelmintics from plant cysteine proteinases. Parasit Vectors 1(1):1–29. CrossRefGoogle Scholar
  10. 10.
    Boomker J, Horak IG, Ramsay KA (1989) Helminth and arthropod parasites of indigenous goats in the Northern Transvaal. Onderstepoort 61:13–20Google Scholar
  11. 11.
    Brophy PM, Patterson LH, Pritchard DL (1995) Secretory nematode SOD–offensive or defensive? Int J Parasitol 25(7):856–865CrossRefGoogle Scholar
  12. 12.
    Buege JA, Aust SD (1978) Microsomal lipid peroxidation. Methods Enzymol 52:302–310CrossRefGoogle Scholar
  13. 13.
    Caito SW, Aschner M (2015) Quantification of Glutathione in Caenorhabditis elegans. Curr Protoc Toxicol 64:6–18. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Chisholm AD, Xu S (2012) The Caenorhabditis elegans epidermis as a model skin II: differentiation and physiological roles. Wiley Interdiscip Rev Dev Biol 1(6):879–902. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Chitra K, Annadurai G (2013) Antimicrobial activity of wet chemically engineered spherical shaped ZnO nanoparticles on food borne pathogen. Int Food Res J 20(1):59–64Google Scholar
  16. 16.
    Chiumiento L, Bruschi F (2009) Enzymatic antioxidant systems in helminth parasites. Parasitol Res 105(3):593–603. CrossRefPubMedGoogle Scholar
  17. 17.
    Citiulo F, Jacobsen ID, Miramón P, Schild L, Brunke S, Zipfel P, Brock M, Hube B, Wilson D (2012) Candida albicans scavenges host zinc via Pra1 during endothelial invasion. PLoS Pathog 8:e1002777. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Corning S (2009) Equine cyathostomins: a review of biology, clinical significance and therapy. Parasit Vectors 2(Suppl 2):S1. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Cox GN, Kusch M, Edgar RS (1981) Cuticle of Caenorhabditis elegans: its isolation and partial characterization. J Cell Biol 90(1):7–17CrossRefGoogle Scholar
  20. 20.
    Craig TM, Diamond PL, Ferwerda NS, Thompson JA (2007) Evidence of ivermectin resistance by Parascaris equorum on a Texas horse farm. J equine vet sci 27(2):67–71CrossRefGoogle Scholar
  21. 21.
    Danikowski KM, Cheng T (2019) Colorimetric analysis of alkaline phosphatase activity in S. aureus Biofilm. J Vis Exp. CrossRefPubMedGoogle Scholar
  22. 22.
    Dorostkar A, Ghalavand M, Nazarizadeh A, Tat M, Hashemzadeh SM (2017) Anthelmintic effects of zinc oxide and iron oxide nanoparticles against Toxocara vitulorum. Int Nano Lett 7(2):157–164. CrossRefGoogle Scholar
  23. 23.
    Doumas BT, Watson WA, Biggs HG (1971) Albumin standards and the measurement of serum albumin with bromcresol green. Clin Chem Acta 31:87–96CrossRefGoogle Scholar
  24. 24.
    Esmaeilnejad B, Samiei A, Mirzaei Y, Farhang-Pajuh F (2018) Assessment of oxidative/nitrosative stress biomarkers and DNA damage in Haemonchus contortus, following exposure to zinc oxide nanoparticles. Acta Parasitol 63(3):563–571. CrossRefPubMedGoogle Scholar
  25. 25.
    Fahmy SR, Sayed DA (2017) Toxicological perturbations of zinc oxide nanoparticles in the Coelatura aegyptiaca mussel. Toxicol Ind Health 33(7):564–575. CrossRefPubMedGoogle Scholar
  26. 26.
    Gandhi PR, Jayaseelan C, Mary RR, Mathivanan D, Suseem SR (2017) Ascaricidal, pediculicidal and larvicidal activity of synthesized ZnO nanoparticles using Momordica charantia leaf extract against blood feeding parasites. Exp Parasitol 181:47–56. CrossRefPubMedGoogle Scholar
  27. 27.
    Gang SS, Hallem EA (2016) Mechanisms of host seeking by parasitic nematodes. Mol Biochem Parasitol 208(1):23–32. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Gherbawy YA, Shalaby IM, Abd El–sadek MS, Elhariry HM, Banaja AA (2013) The anti–fasciolasis properties of silver nanoparticles produced by Trichoderma harzianum and their improvement of the anti–fasciolasis drug triclabendazole. Int J Mol Sci 14(11):21887–21898. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Gonzalez–Moragas L, Roig A, Laromaine A (2015) C. elegans as a tool for in vivo nanoparticle assessment. Adv Colloid Interface Sci 219:10–26. CrossRefPubMedGoogle Scholar
  30. 30.
    Gopalakrishnan K, Ramesh C, Ragunathan V, Thamilselvan M (2012) Antibacterial activity of Cu2O nanoparticles on E. coli synthesized from Tridax procumbens leaf extract and surface coating with polyaniline. Dig J Nanomater Bios 7:833–839Google Scholar
  31. 31.
    Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione S–transferases; the first enzymatic step in mercapturic acid formation. J Biol Chem 249:7130–7139Google Scholar
  32. 32.
    Henry RJ (1964) Colorimetric determination of total protein. In: clinical chemistry. Harper and Row Publisher, New York, p 181Google Scholar
  33. 33.
    Hewitson JP, Grainger JR, Maizels RM (2009) Helminth immunoregulation: the role of parasite secreted proteins in modulating host immunity. Mol Biochem Parasitol 167(1):1–11. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Kalpana VN, Devi Rajeswari V (2018) A review on green synthesis, biomedical applications, and toxicity studies of ZnO NPs. Bioinorg Chem Appl 2018:3569758. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Khan YA, Singh BR, Ullah R, Shoeb M, Naqv AH, Abidi SM (2015) Anthelmintic effect of biocompatible zinc oxide nanoparticles (ZnO NPs) on Gigantocotyle explanatum, a neglected parasite of Indian water buffalo. PLoS One 10(7):e0133086. CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Knox DP, Kennedy MW (1988) Proteinases released by the parasitic larval stages of Ascaris suum, and their inhibition by antibody. Mol Biochem Parasitol 28:207–216CrossRefGoogle Scholar
  37. 37.
    Kotze AC (2003) Catalase induction protects Haemonchus contortus against hydrogen peroxide in vitro. Int J Parasitol 33:393–400CrossRefGoogle Scholar
  38. 38.
    Kotze AC, Clifford S, O’Grady J, Behnke JM, McCarthy JS (2004) An in vitro larval motility assay to determine anthelmintic sensitivity for human hookworm and Strongyloides species. Am J Trop Med Hyg 71(5):608–816CrossRefGoogle Scholar
  39. 39.
    Leslie JF, Cain GD, Meffe GK, Vrijenhoek RC (1982) Enzyme polymorphismin Ascaris suum (Nematoda). J Parasitol 68(4):576–587CrossRefGoogle Scholar
  40. 40.
    Li YX, Wang Y, Hu YO, Zhong JX, Wang DY (2011) Modulation of the assay system for the sensory integration of 2 sensory stimuli that inhibit each other in nematode Caenorhabditis elegans. Neurosci Bull 27(2):69–82. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Liu RM, Gaston Pravia KA (2010) Oxidative stress and glutathione in TGF-β-mediated fibrogenesis. Free Radic Biol Med 48(1):1–15. CrossRefPubMedGoogle Scholar
  42. 42.
    Mart´inez A (1995) Nitric oxide synthase in invertebrates. Histochem J 27(10):770–776CrossRefGoogle Scholar
  43. 43.
    Masetti M, Locci T, Cecchettini A, Lucchesi P, Magi M, Malvaldi G, Bruschi F (2004) Nitric oxide synthase immunoreactivity in the nematode Trichinella britovi. Evidence for nitric oxide production by the parasite. Int J Parasitol 34(6):715–721. CrossRefPubMedGoogle Scholar
  44. 44.
    Minning DM, Gow AJ, Bonaventura J, Braun R, Dewhirst M, Goldberg DE, Stamler JS (1999) Ascaris haemoglobin is a nitric oxide-activated deoxygenase. Nature 401(6752):497–502. CrossRefPubMedGoogle Scholar
  45. 45.
    Morsy K, Bashtar A, Al Quraishy S, Adel S (2016) Description of two equine nematodes, Parascaris equorum Goeze 1782 and Habronema microstoma Schneider 1866 from the domestic horse Equus ferus caballus (Famisly: Equidae) in Egypt. Parasitol Res 115:4299–4306CrossRefGoogle Scholar
  46. 46.
    Murdock RC, Braydich-Stolle L, Schrand AM, Schlager JJ, Hussain SM (2007) Characterization of nanomaterial dispersion in solution prior to in vitro exposure using dynamic light scattering technique. Toxicol Sci 101(2):239–253. CrossRefPubMedGoogle Scholar
  47. 47.
    Nandurkar HP, Zambare SP (2012) Comparative study of acute and chronic exposure of chloramphenicol on total lipid contents in different tissues of model animals, Lamellidens corrianus (Lea) and Parreysia cylindrica (Annandale and Prashad). Int J Multidiscip Res 2(3):33–35Google Scholar
  48. 48.
    Nazarizadeh A, Asri-Rezaie S (2016) Comparative study of antidiabetic activity and oxidative stress induced by zinc oxide nanoparticles and zinc sulfate in diabetic rats. AAPS Pharm Sci Tech 17(4):834–843. CrossRefGoogle Scholar
  49. 49.
    Oberdörster G, Oberdörster E, Oberdörster J (2005) Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 113(7):823–839. CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Patterson AL (1939) The Scherrer formula for X-ray particle size determination. Phys Rev 56:978. CrossRefGoogle Scholar
  51. 51.
    Premanathan M, Karthikeyan K, Jeyasubramanian K, Manivannan G (2011) Selective toxicity of ZnO nanoparticles toward Gram-positive bacteria and cancer cells by apoptosis through lipid peroxidation. Nanomed Nanotechnol Biol Med 7(2):184–192CrossRefGoogle Scholar
  52. 52.
    Rashid MM, Ferdous J, Banik S, Islam MR, Uddin AH, Robel FN (2016) Anthelmintic activity of silver-extract nanoparticles synthesized from the combination of silvernanoparticles and M. charantia fruit extract. BMC Complement Altern Med 16:242CrossRefGoogle Scholar
  53. 53.
    Reitman S, Frankel S (1957) A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. Am J Clin Pathol 28(1):56–63CrossRefGoogle Scholar
  54. 54.
    Richmond W (1973) Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum. Clin Chem 19(12):1350–1356PubMedGoogle Scholar
  55. 55.
    Rizvi A, Hasan S, Alam M, Zafar A, Fatima T, Shareef PA, Banu N, Saleemuddin M, Saifullah MK, Abidi SM (2012) Levels of some antioxidant molecules and lipid peroxidation during in vivo transformation of the progenetic metacercaria of Clinostomum complanatum to ovigerous adult worms. Vet Parasitol 185(2–4):164–167. CrossRefPubMedGoogle Scholar
  56. 56.
    Sakr S, Al Lail SM (2005) Fenvalerate induced histopathological and histochemical changes in the liver of the cat fish Clarias gariepinus. J Appl Sci Res 1:263–267Google Scholar
  57. 57.
    Shafaei S, Al Farahnak, Golmohammadi T, Esharghian MR, Rad MBM (2014) Study of Triclabendazole (TCBZ) effect on Aspartate Transaminase (AST) activity of Fasciola gigantica parasite and liver enzyme activity assay. J Pharma care 2(4):149–153Google Scholar
  58. 58.
    Shalaby HA (2013) Anthelmintics resistance; how to overcome it? Iran J Parasitol 8(1):18–32PubMedPubMedCentralGoogle Scholar
  59. 59.
    Shoeb M, Singh BR, Khan JA, Khan W, Singh BN, Singh HB, Singh HB, Naqvi AH (2013) ROS-dependent anticandidal activity of zinc oxide nanoparticles synthesized by using egg albumen as a biotemplate. Adv Nat Sci Nanosci Nanotechnol 4:1–11. CrossRefGoogle Scholar
  60. 60.
    Smith NC, Bryant C (1989) The effects of antioxidants on the rejection of Nippostrongylus brasiliensis. Parasite Immunol 11(2):161–167CrossRefGoogle Scholar
  61. 61.
    Song S, Guo Y, Zhang X, Zhang X, Zhang J, Ma E (2014) Changes to cuticle surface ultrastructure and some biological functions in the nematode Caenorhabditis elegans exposed to excessive copper. Arch Environ Contam Toxicol 66(3):390–399. CrossRefPubMedGoogle Scholar
  62. 62.
    Starnes D, Unrine J, Chen C, Lichtenberg S, Starnes C, Svendsen C, Kille P, Morgan J, Baddar ZE, Spear A, Bertsch P, Chen KC, Tsyusko O (2019) Toxicogenomic responses of Caenorhabditis elegans to pristine and transformed zinc oxide nanoparticles. Environ Pollut 247:917–926. CrossRefPubMedGoogle Scholar
  63. 63.
    Stein EA, Myers G (1994) Lipids, lipoproteins and apolipoproteins. In: Burtis CA, Ashwood ER (eds) Tietz textbook of clinical chemistry. WB Saunders Company, Philadelphia, pp 1002–1993Google Scholar
  64. 64.
    Tydén E, Dahlberg J, Karlberg O, Höglund J (2014) Deep amplicon sequencing of preselected isolates of Parascaris equorum in β-tubulin codons associated with benzimidazole resistance in other nematodes. Parasit Vectors 29:7–410. CrossRefGoogle Scholar
  65. 65.
    Wang J, Dai H, Nie Y, Wang M, Yang Z, Cheng L, Liu Y, Chen S, Zhao G, Wu L, Guang S, Xu A (2018) TiO2 nanoparticles enhance bioaccumulation and toxicity of heavy metals in Caenorhabditis elegans via modification of local concentrations during the sedimentation process. Ecotoxicol Environ Saf 162:160–169. CrossRefPubMedGoogle Scholar
  66. 66.
    Wang N, Wang H, Tang C, Lei S, Shen W, Wang C, Wang G, Wang Z, Wang L (2017) Toxicity evaluation of boron nitride nanospheres and water-soluble boron nitride in Caenorhabditis elegans. Int J Nanomed 8(12):5941–5957. CrossRefGoogle Scholar
  67. 67.
    Wang Q, Rosa BA, Jasmer DP, Mitreva M (2015) Pan-Nematoda transcriptomic elucidation of essential intestinal functions and therapeutic targets with broad potential. EBioMedicine 2(9):1079–1089. CrossRefPubMedPubMedCentralGoogle Scholar
  68. 68.
    Xiong D, Fang T, Yu L, Sima X, Zhu W (2011) Effects of nano-scale TiO2, ZnO and their bulk counterparts on zebrafish: acute toxicity, oxidative stress and oxidative damage. Sci Total Environ 409(8):1444–1452. CrossRefPubMedGoogle Scholar

Copyright information

© Witold Stefański Institute of Parasitology, Polish Academy of Sciences 2019

Authors and Affiliations

  1. 1.Biology DepartmentCollege of Science, King Khalid UniversityAbhaSaudi Arabia
  2. 2.Zoology Department, Faculty of ScienceCairo UniversityGizaEgypt

Personalised recommendations