Anticryptosporidial effect of pomegranate peels water extract in experimentally infected mice with special reference to some biochemical parameters and antioxidant activity

  • D. AboelsouedEmail author
  • F. A. M. Abo-Aziza
  • M. H. Mahmoud
  • K. N. Abdel Megeed
  • N. M. T. Abu El Ezz
  • F. M. Abu-Salem
Original Article


Cryptosporidiosis is a zoonotic disease caused by a well-known parasitic protozoan called Cryptosporidium. Infection in livestock causes important economic losses among farm animals and its control has a global concern. In this study, internal white and external red layers were separated from pomegranate peels (Punica granatum) then; they were grinded to reach Nano form. Anticryptosporidial effect of their water extracts was investigated in experimentally infected mice. Also, their antioxidant activity, biochemical and histopathological changes were studied. Briefly, hot aqueous extracts of pomegranate peels were prepared regarding its good sensory attributes at concentration of 10% W/V. Analysis of total phenolics, individual phenolics by HPLC-DAD and antioxidant activities have been done. Forty-five mice were divided into five groups each one containing nine mice. The first group was healthy mice and the 2nd one was infected orally with 104Cryptosporidium parvum (C. parvum) oocysts/mice and not treated. The other 3 groups were infected and orally treated with Nitazoxanide (NTZ) for the 3rd group, pomegranate red peel extract for the 4th group and pomegranate white peel extract for the 5th group. Blood samples were collected after 1 and 2 weeks post treatment for protein profile, liver enzymes and antioxidant activity evaluation. After 3 weeks, all animals were sacrificed and ileal tissues were embedded in paraffin for histopathological examination. The results showed that pomegranate peel extracts were rich in phenolic compounds, had high antioxidant activity and therapeutic effect on C. parvum in experimentally infected mice. Red peel extract diminished C. parvum oocysts count significantly in experimentally infected mice than white peel and NTZ treatments. Also, the histopathological examination revealed that red peel treated mice ileal sections showed a great enhancement in the shape and structure of villi towards normal structure than other treated groups. Most of the measured biochemical parameters after 2 weeks’ treatment with red pomegranate peel and NTZ were enhanced in their concentrations towards the healthy normal status. In conclusion, this study showed the effectiveness of Nano-form of pomegranate white and red peel extracts against C. parvum oocysts. Pomegranate red peel extract was found to have antioxidant activity that could significantly enhance the serum biochemical parameters and oxidative stress towards the healthy normal status. Furthermore, it is suggested that pomegranate peel should be separated and used in the daily animal diet or as a functional beavarage for human as accepted from the panelists to give protective effects against this parasite as well as to improve health benefits.


Cryptosporidium parvum Therapeutic effect Pomegranate peel Nano-form Antiparasitic beverage Biochemical parameters Antioxidant activity 


Author contributions statement

All authors prepared the research plan and the design of experiments. Aboelsoued D. separated C. parvum oocysts from calf fecal samples, carried out the experimental infection in mice, administrated the therapeutic doses, collected mice fecal pellets everyday and counted oocysts in feces throughout the experiment. Abo-Aziza F. collected the blood samples from mice, separated sera and carried out the biochemical analysis including protein profile, liver enzymes and serum antioxidant activity. Mahmoud M. prepared pomegranate peel powder by milling to achieve Nano-form, determination of proximate composition, droplet size measurement, total phenolics and polyphenols and antioxidant activities. Also, beverage preparation and sensory evaluation. Abdel Megeed K. and Abu El Ezz N. participated in dose preparation and sacrificing of mice. Abu El Ezz N, Abdel Megeed K. and Aboelsoued D. examined stained ileal sections and interpreted them histologically and pathologically. Aboelsoued D, Abo-Aziza F and Mahmoud M analyzed, interpreted the data and prepared the manuscript. All authors had critically read and approved the final manuscript.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflicts or competing interests.


  1. Abdel-Ghaffar F, Semmler M, Al-Rasheid KA, Strassen B, Fischer K, Aksu G, Klimpel S, Mehlhorn H (2010) The effects of different plant extracts on intestinal cestodes and on trematodes. Parasitol Res 108:979–984Google Scholar
  2. Abdel Megeed KN, Hammam AM, Morsy GH, Khalil FAM, Seliem MME, Aboelsoued D (2015) Control of cryptosporidiosis in buffalo calves using garlic (Allium sativum) and Nitazoxanide with special reference to some biochemical parameters. Glob Vet 14(5):646–655Google Scholar
  3. Abdelrahman KA, Abdel Megeed KN, Hammam AM, Morsy GH, Seliem MME, Aboelsoued D (2015) Molecular characterization of bubaline isolate of Cryptosporidium species from Egypt. Res J Parasitol 10(4):127–141Google Scholar
  4. Abdou AG, Harba NM, Afifi AF, Elnaidany NF (2013) Assessment of Cryptosporidium parvum infection in immunocompetent and immunocompromised mice and its role in triggering intestinal dysplasia. Int J Infect Dis 17(8):593–600Google Scholar
  5. Abid M, Yaich H, Cheikhrouhou S, Khemakhem I, Bouaziz M, Attia H, Ayadi MA (2017) Antioxidant properties and phenolic profile characterization by LC–MS/MS of selected Tunisian pomegranate peels. J Food Sci Technol 54(9):2890–2901Google Scholar
  6. Abo-Aziza FAM, Hendawy SHM, Zaki AA, Oda SS, El Namaky AH (2017) Clinicohistopathological and immunological alterations in Egyptian donkeys infested by Rhinoestrus spp. during the winter season. Egypt J Vet Sci 48(2):61–71Google Scholar
  7. Abou-Arab AA, Mahmoud MH, Abu-Salem FM (2016) Bioactive compounds content of citrus peel as affected by drying processes. Inter J Biol Biomol Agricul Food Biotech Eng 10(4):225–228Google Scholar
  8. Abu El Ezz NMT, Khalil AM, Shaapan RM (2011) Therapeutic effect of onion (Allium cepa) and cinnamon (Cinnamomum zeylanicum) oils on Cryptosporidiosis in experimentally infected mice. Glob Vet 7(2):179–183Google Scholar
  9. Al-Mathal EM, Alsalem AA (2012) Pomegranate (Punica granatum) peel is effective in a murine model of experimental Cryptosporidium parvum. Exp Parasitol 131:350–357Google Scholar
  10. Al-Mathal EM, Alsalem AA (2013) Pomegranate (Punica granatum) peel is effective in a murine model of experimental Cryptosporidium parvum ultrastructural studies of the ileum. Exp Parasitol 134:482–494Google Scholar
  11. Amer OS, Dkhil MA, Hikal WM, Al-Quraishy S (2015) Antioxidant and anti-inflammatory activities of pomegranate (Punica granatum) on Eimeria papillata-induced infection in mice. BioMed Res Int 2015:219670Google Scholar
  12. Anahita A, Asmah R, Fauziah O (2015) Evaluation of total phenolic content, total antioxidant activity, and antioxidant vitamin composition of pomegranate seed and juice. Gen Med 3(1):1–4Google Scholar
  13. AOAC (2000) Official methods of analysis of the association of official analytical chemists, 17th edn. The Association of Official Analytical Chemists, RockvilleGoogle Scholar
  14. Azza MK (2008) Some biochemical, hematological and clinical studies of selected ruminal and blood constituents in camels affected by various diseases. Res J Vet Sci 1(1):16–27Google Scholar
  15. Bancroft JD, Stevens GA (1990) Theory and practice of histological techniques, 2nd edn. Churchill Livingstone, LondonGoogle Scholar
  16. Barros HRM, Ferreira TAPC, Genovese MI (2012) Antioxidant capacity and mineral content of pulp and peel from commercial cultivars of citrus from Brazil. Food Chem 134:1892–1898Google Scholar
  17. Cannon DC, Olitzky I, Inkpen JA (1974) Clinical chemistry principles and techniques of determination of total protein, 2nd edn. Harper and Rowpubl, LondonGoogle Scholar
  18. Chalmers RM, Davies AP (2010) Minireview: clinical cryptosporidiosis. Exp Parasitol 124:138–146Google Scholar
  19. Chaudhry Q, Castle L (2011) Food applications of nanotechnologies: an overview of opportunities and challenges for developing countries. Trends Food Sci Tech 22:1–9Google Scholar
  20. Chidambara Murthy KN, Jayaprakasha GK, Singh RP (2002) Studies on antioxidant activity of pomegranate (Punica granatum) peel extract using in vivo models. J Agric Food Chem 50:4791–4795Google Scholar
  21. Current WL, Reese NC (1986) A comparison of endogenous development of three isolates of Cryptosporidium in suckling mice. J Protozool 33:98–108Google Scholar
  22. Dell’Agli M, Galli G, Corbett Y, Taramelli D, Lucantoni L, Habluetzel A, Maschi O, Caruso D, Giavarini F, Romeo S, Bhattacharya D, Bosisio E (2009) Antiplasmodial activity of Punica granatum L. fruit rind. J Ethnopharmacol 125:279–285Google Scholar
  23. Dkhil MA (2013) Anticoccidial, anthelmintic and antioxidant activities of pomegranate (Punica granatum) peel extract. Parasitol Res 112:2639–2646Google Scholar
  24. Doumas B, Watson W, Biggs M (1971) Albumin standards and the measurement of serum albumin with bromocresyl green. Clin Chim Acta 31:87–96Google Scholar
  25. El Mageid MMA, Salama NA, Saleh MAM, Abo-Taleb HM (2016) Evaluation of antdiabetic, hypocholesterolemic of pomegranate (Punica granatum L.) juice powders and peel powder extracts in male albino rats. IOSR-JPBS 11(6):53–64Google Scholar
  26. Enwezor FNC, Sackey AKB (2005) Camel trypanosomosis—a review. Vet Arh 75:439–452Google Scholar
  27. Han J, Weng X, Bi K (2008) Antioxidants from a Chinese Medicinal Herb-Lithospermum erythrorhizon. Food Chem 106(1):2–10Google Scholar
  28. Hassanain MA, Khalil FAM, Abd El-Razik KA, Shaapan RM (2011) Prevalence and molecular discrimination of Cryptosporidium parvum in calves in Behira provinces, Egypt. Res J Parasitol 6:101–108Google Scholar
  29. Hayat K, Zhang X, Farooq U, Abbas S, Xia S, Jia C, Zhong F, Zhang J (2010) Effect of microwave treatment on phenolic content and antioxidant activity of citrus mandarin pomace. Food Chem 123:423–429Google Scholar
  30. Hegazi AG, Abdel Megeed KN, Hassan SE, Abdelaziz MM, Toaleb NI, El Shanawany EE, Aboelsoued D (2018) Comparative ovicidal activity of Moringa oleifera leaf extracts on Fasciola gigantica eggs. Vet World 11(2):215–220Google Scholar
  31. Henriksen SA, Pohlenz JF (1981) Staining of cryptosporidia by a modified Ziehl-Neelsen technique. Acta Vet Scand 22:594–596Google Scholar
  32. Hilali M, Abdel-Gawad A, Nassar A, Abdel-Wahab A (2006) Hematological and biochemical changes in water buffalo calves (Bubalus bubalis) infected with Trypanosoma evansi. Vet Parasitol 139:237–243Google Scholar
  33. Hosseini A, Jafari SM, Mirzaei H, Asghari A, Akhavan S (2015) Application of image processing to assess emulsion stability and emulsification properties of Arabic gum. Carbohydr Polym 126:1–8Google Scholar
  34. John J, Mehta A, Shukla S, Mehta P (2009) A report on anthelmintic activity of Cassia tora leaves. J Sci Technol 31(3):269–271Google Scholar
  35. Kharchoufi S, Licciardello F, Siracusa L, Muratore G, Hamdi M, Restuccia C (2018) Antimicrobial and antioxidant features of ‘Gabsiʼ pomegranate peel extracts. Ind Crops Prod 11:345–352Google Scholar
  36. Lacroix S, Mancassola R, Naciri M, Laurent F (2001) Cryptosporidium parvum-specific mucosal immune response in C57BL/6 neonatal and gamma interferon-deficient mice: role of tumor necrosis factor alpha in protection. Infect Immun 69:42–1635Google Scholar
  37. Li Y, Guo C, Yang J, Wei J, Xu J, Cheng S (2006) Evaluation of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract. Food Chem 96(2):254–260Google Scholar
  38. Lockman PR, Mumper RJ, Khan MA, Allen DD (2002) Nanoparticle technology for drug delivery across the blood brain barrier. Drug Dev Ind Pharm 28:1–13Google Scholar
  39. Mahmoud MH, Seleet FL, Foda MI (2017) Effect of different concentration techniques in some properties of fresh and storage Pomegranate juice. Asian J Sci Res 10(4):290–298Google Scholar
  40. Mahmoud MH, Wahba HM, Mahmoud MH, Abu-Salem FM (2018) Antagonizing the hazardous impact of increased oxidative stress in Wistar rats by biscuits with dried orange peel. J Biol Sci 18(1):21–31Google Scholar
  41. Malina JM, Rodriguez-ponce E, Ferrer O, Cutierrez AC, Herndez S (1994) Biopathological data of goat kids with cryptosporidiosis. Vet Rec 135:67–68Google Scholar
  42. Matsui T, Fujino T, Kajima J, Tsuji M (2001) Infectivity and oocyst excretion patterns of Cryptosporidium muris in slightly infected mice. J Vet Med Sci 63:20–319Google Scholar
  43. Mead JR, Nurcan I, Xiangdong Y, Yelena B, Michael J, Michael TF, Raymond FS (1994) Infection dynamics and clinical features of cryptosporidiosis in SCID mice. Infect Immun 62(5):1691–1695Google Scholar
  44. Moneim AEA (2012) Antioxidant activities of Punica granatum (pomegranate) peel extract on brain of rats. J Med Plants Res 6(2):195–199Google Scholar
  45. Morsy GH, Megeed KNA, Hammam AM, Seliem MME, Khalil FAM, Aboelsoued D (2014) Prevalence of Cryptosporidium infection in buffalo calves with special reference to urea and creatinine levels. Glob Vet 13(5):662–667Google Scholar
  46. Morsy M, Mekawi E, Elsabagh R (2018) Impact of pomegranate peel nanoparticles on quality attributes of meatballs during refrigerated storage. LWT 89:489–495Google Scholar
  47. Mousa N, Ahmed A, El-Nahas H, Atef E, Mohammad A, Nabih M, Magdy H, Mohammad E, Narmin E, Walled E (2014) Cryptosporidiosis in patients with diarrhea and chronic liver diseases. J Infect Dev Ctries 8(12):1584–1590Google Scholar
  48. Nawwar MAM, Hussein SAM, Merfort I (1994) Leaf phenolics of Punica granatum. Phytochemistry 37:1175–1177Google Scholar
  49. Oyaizu M (1986) Studies on antioxidative activities of browning reaction products prepared from glucosamine. Jpn J Nutr 44:307–315Google Scholar
  50. Petrie A, Watson P (1999) Statistics for veterinary and animal science, 1st edn. The Blackwell Sci. Ltd, Oxford, pp 90–99Google Scholar
  51. Pumipuntu N, Piratae S (2018) Cryptosporidiosis: a zoonotic disease concern. Vet World 11(5):681–686Google Scholar
  52. Qnais EY, Elokda AS, Abu Ghalyun YY, Abdulla FA (2007) Antidiarrheal activity of the aqueous extract of Punica granatum (pomegranate) peels. Pharm Biol 45(9):715–720Google Scholar
  53. Ramful D, Tarnus E, Aruoma OI, Bourdon E, Bahorun T (2011) Polyphenol composition, vitamin C content and antioxidant capacity of Mauritian citrus fruit pulps. Food Res Inter 44:2088–2099Google Scholar
  54. Saleh MA, Mahran OM, Al-Salahy BM (2011) Circulating oxidative stress status in dromedary camels infested with sarcoptic mange. Vet Res Commun 35(1):35–45Google Scholar
  55. Sasahara T, Maruyama H, Aoki M, Kikuno R, Sekiguchi T, Takahashi A, Satoh Y, Kitasato H, Takayama Y, Inoue M (2003) Apoptosis of intestinal crypt epithelium after Cryptosporidium parvum infection. J Infect Chemother 9(3):278–281Google Scholar
  56. Seeram NP, Schulman RN, Heber D (2006) Pomegranates: ancient roots to modern medicine. Taylor and Francis Group, Boca Raton, pp 5–8Google Scholar
  57. Shafiq MAB, Maqbool A, Khan UJ, Lateef M, Ijaz M (2015) Prevalence, water borne transmission and chemotherapy of cryptosporidiosis in small ruminants. Pak J Zool 47(6):1715–1721Google Scholar
  58. Shiban MS, Al-Otaibi MM, Al-Zoreky NS (2012) Antioxidant activity of pomegranate (Punica granatum L.) fruit peels. Food Nutr Sci 3:991–996Google Scholar
  59. Singh B, Singh JP, Kaur A, Singh N (2018) Phenolic compounds as beneficial phytochemicals in pomegranate (Punica granatum L.) peel: a review. Food Chem 261:75–86Google Scholar
  60. Snedecor GW, Cochran WG (1980) Statistical methods, 7th edn. Iowa State University Press, AmesGoogle Scholar
  61. Tanaka T, Nonaka GI, Nishioka I (1986) Tannins and related compounds. XLI. Isolation and characterization of novel ellagitannins, punicacorteins A, B, C and D and punigluconin from the bark of Punica granatum L. Chem Pharm Bull 34:656–663Google Scholar
  62. Ventura J, Alarcón-Aguilar F, Roman-Ramos R, Campos-Sepulveda E, Reyes-Vega ML, Boone-Villa VD, Jasso-Villagómez EI, Aguilar CN (2013) Quality and antioxidant properties of a reduced-sugar pomegranate juice jelly with an aqueous extract of pomegranate peels. Food Chem 136:109–115Google Scholar
  63. Wang R, Ding Y, Liu R, Xiang L, Du L (2010) Pomegranate: constituents, bioactivities and pharmacokinetics. Fruit Veg Cereal Sci Biotechnol 4:77–87Google Scholar
  64. Winn-Deen ES, David H, Sigler G, Chavez R (1988) Determination of total and pancreatic α-amylase in human serum with 2-chloro-4-nitrophenyl-α-d-maltotrioside as substrate. Clin Chem 34:2005Google Scholar
  65. Zohreh K, Parvaneh K, Solmaz C, Soheila KN (2012) Comparative study of serum lipid profile in chicken, ostrich, cattle and sheep. Comp Clin Pathol 21:259–263Google Scholar

Copyright information

© Indian Society for Parasitology 2019

Authors and Affiliations

  1. 1.Department of Parasitology and Animal DiseasesNational Research CentreDokkiEgypt
  2. 2.Department of Food TechnologyNational Research CentreDokkiEgypt

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