In vitro Study of Effects of Alcoholic Extract of Pomegranate Peel on Ichthyophthirius multifiliis Theronts

Document Type : Research Paper

Authors

1 Department of Aquatic Animal Health, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

2 Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

3 Department of Pathobiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

Abstract

The Ichthyophthirius multifiliis (Ich) is one of the most important parasites in aquaculture industry, which causes the fatal disease of Ichthyophthiriasis. Treatments for this parasite include the use of certain chemical medications. However, due to the many negative effects of chemical compounds on the environment and humans, extensive studies of plant extract effectiveness in treating this parasite are very important. This study evaluated the alcoholic pomegranate (Punica granatum) peel extract's antiparasitic activity against the parasite I. multifiliis in vitro. Under laboratory conditions, the anti-parasitic efficacy of pomegranate peel alcoholic extract against I. multifiliis at doses of (0.5, 1, 2, 4, and 8 g/l) was evaluated after exposure for six hours. Additionally, this extract's toxicity was assessed for 96 hours on zebrafish at dosages of 0.5, 1, 2 and 4 g/liter. The collected data were statistically contrasted with the results of the positive control sample (15 ppm formalin) and the negative control treatment. The study revealed that the theronts may be destroyed in 6 hours by concentrations of 2, 4, and 8 g/liter, in addition to the concentration of 1 g/liter, which caused the death of 96% of theronts within this period. In the toxicity test, the concentrations (2, 4, and 8 g/L) were highly toxic, and all the fish died, but the concentrations of 1 and 0.5 g/L were safe doses within 96 hours. Also, the value of EC50 in this research was calculated as 1.41 g/liter. Therefore, the alcoholic extract of pomegranate peel in doses of 2, 4, and 8 g/l is not effective for clinical treatment and is only suitable as an antiseptic. So, the dose of 1 g/l has very good results and is recommended for clinical treatment.

Keywords

Main Subjects


  1. Sasani F., Shokrpoor S., Rahmati-Holasoo H., Zargar A. Appearance of Red Mark Syndrome (RMS) in cultured rainbow trout (Oncorhynchus mykiss Walbaum, 1972) in Iran. Bulletin of European Association of Fish Pathologists. 2016; 36(2): 90-94.
  2. Ahmadivand S., Weidmann M., El-Matbouli M., Rahmati-Holasoo H. Low pathogenic strain of infectious pancreatic necrosis virus (IPNV) associated with recent outbreaks in Iranian trout farms. Pathogens. 2020; 9(10): 782.
  3. Seyrafi R., Najafi G., Rahmati-Holasoo H., Hajimohammadi B., Shamsadin A.S. Histological study of hepatopancreas in iridescent shark catfish (Pangasius hypophthalmus). J. Animal and Veterinary Advances. 2009; 8(7): 1305-1307.
  4. Rahmati Holasoo H., Marandi A., Ebrahimzadeh Mousavi H., Azizi A. Study of the losses of Siberian sturgeon (Acipenser baerii) due to gill infection with Diclybothrium armatum in sturgeon farms of Qom and Mazandaran provinces. J. Animal Environment 2021; 13(4): 193-200.
  5. Rahmati-Holasoo H., Marandi A., Ebrahimzadeh Mousavi H., Taheri Mirghaed A. Parasitic fauna of farmed freshwater ornamental fish in the northwest of Iran. Aquaculture International. 2022; 30, 633-652.
  6. Rahmati‐Holasoo H., Tavakkoli S., Ebrahimzadeh Mousavi H., Marandi A., Taheri Mirghaed, A. Parasitic fauna of farmed freshwater ornamental sutchi catfish (Pangasiandon hypophthalmus) and silver dollar (Metynnis hypsauchen) in Alborz province, Iran. Veterinary Medicine and Science. 2023; 9(4): 1627-1635.
  7. Rahmati-Holasoo H., Niyyati M., Fatemi M., Mahdavi Abhari F., Shokrpoor S., Nassiri A., Marandi, A. Molecular identification, phylogenetic analysis and histopathological study of pathogenic free-living amoebae isolated from discus fish (Symphysodon aequifasciatus) in Iran: 2020-2022. BMC Veterinary Research. 2024; 20(1): 54.
  8. Canella M.F., Rocchi-Canella I. Biologie des Ophryoglenina (cilies hymenostermes, histophages). Annals of the University of Ferrara (N.S. Sect. 111). 1976; 3 (Suppl. 2), 1-510.
  9. Corliss J. The Ciliated Protozoa: Characterization, Classification, and Guide to the Literature. Pergamon Press, Oxford, UK. 1979.
  10. Wright A.D., Lynn D.H. Phylogeny of the fish parasite Ichthyophthirius and its relatives Ophryoglena and Tetrahymena (Ciliophora, Hymenostomatia) inferred from 18s ribosomal RNA sequences. Molecular and Biological Evolution. 1995; 12(2): 285-290.
  11. Lin T.L., Clark T.G., Dickerson, H. Passive immunization of channel catfish (Ictalurus punctatus) against the ciliated protozoan parasite Ichthyophthirius multifiliis by use of murine monoclonal antibodies. Infection and Immunity. 1996; 64: 4085-4090. https://doi.org/10.1128/iai.64.10.4085-4090.1996
  12. Van Den Bussche R.A., Hoofer S.R., Drew C.P., Ewing, M.S. Characterization of histone h3/h4 gene region and phylogenetic affinity of Ichthyophthirius multifiliis based on h4 DNA sequence variation. Molecular and Phylogenetic Evolution. 2000; 14(3): 461-468.
  13. Wurtsbaugh W.A. Tapia R.A. Mass mortality of fishes in Lake Titicaca (Peru-Bolivia) associated with the protozoan parasite Ichthyophthirius multifiliis. Transactions of the American Fisheries Society. 1988; 117(2):213-217.
  14. Aguilar A., Alvarez M., Leiro J., Sanmartin M. Parasite populations of the European eel (Anguilla anguilla L.) in the Rivers Ulla and Tea (Galicia, northwest Spain). Aquaculture. 2005; 249 (1), 85-94. https://doi.org/10.1016/j.aquaculture.2005.04.052
  15. Matthews R.A. Ichthyophthirius multifiliis Fouquet and ichthyophthiriosis in freshwater teleosts. Advances in Parasitology. 2005; 59: 159-241.
  16. Maceda-Veiga A., Salvado H., Vinyoles, D., De Sostoa, A. Outbreaks of Ichthyophthirius multifiliis in redtail barbs Barbus haasi in a Mediterranean stream during drought. J. Aquatic Animal Health. 2009; 21: 189-194. 1
  17. Forwood J.M., Harris J.O., Landos M., Deveney, M. R. Life cycle and settlement of an Australian isolate of Ichthyophthirius multifiliis Fouquet, 1876 from rainbow trout. Folia Parasitologica. 2015; 62, 013.
  18. Rintamäki-Kinnunen P., Valtonen E.T. Epizootiology of protozoans in farmed salmonids at northern latitudes. International J. Parasitology. 1997;27:89-99.
  19. Dickerson H.W. Ichthyophthirius multifiliis and Cryptocaryon irritans (phylum Ciliophora). In: Woo, P.T.K. (ed.) Fish Diseases and Disorders. Volume 1. Protozoan and Metazoan Infections, 2nd edn. CABI Publishing, Wallingford, Oxon, UK. 2006; Pp 181-227.
  20. Hines R.S., Spira D.T. Ichthyophthirius multifiliis (Fouquet) in the mirror carp, Cyprinus carpio L. I. Course of infection. J. Fish Biology. 1973; 5: 385-392.
  21. Buchmann K., Sigh J., Nielsen C.V., Dalgaard M. Host responses against the fish parasitizing ciliate Ichthyophthirius multifiliis. Veterinary Parasitology. 2001; 100,105-116.
  22. Tieman D. M., Goodwin A. E. Treatments for ich infestations in channel catfish evaluated under static and flow-through water conditions. N. Am. J. Aquaculture. 2001; 63: 293-299. https://doi.org/10.1577/1548-8454(2001)063<0293:TFIIIC>2.0.CO;2
  23. Murray A. G., Peele E. J. A framework for understanding the potential for emerging diseases in aquaculture. Preventive Veterinary Medicine. 2005; 67: 223‐235.
  24. Sanabria C., Diamant A., Zilberg D. Effects of commonly used disinfectants and temperature on swim bladder non-inflation in freshwater angelfish, Pterophyllum scalare (Lichtenstein). Aquaculture. 2009; 293: (3-4), 158-165.
  25. Buchmann K., Jensen P.B., Kruse K.D. Effects of sodium percar-bonate and garlic extract on Ichthyophthirius multifiliis theronts and tomocysts: In vitro experiments. North American J. Aquaculture. 2003; 65 (1): 21–24.
  26. Ekanem A.P., Obiekezie A., Kloas W., Knopf K. Effects of crude extracts of Mucuna pruriens (Fabacea) and Carica papaya (Caricaceae) against the protozoan fish parasite Ichthyophthirius multifiliis. Parasitology Research. 2004; 92(5): 361-366.
  27. Yao J.Y., Shen J.Y., Li X.L., Xu Y., Hao G.J., Pan X.Y., Wang G.X., Yin W.L. Effect of Effect of sanguinarine from the leaves of Macleaya cordata against Ichthyophthirius multifiliis in grass carp (Ctenopharyngodon idella). Parasitology Research. 2010;107:1035-1042.
  28. Gholipour-Kanani H., Sahandi J., Taheri A. Influence of garlic (Allium sativum) and mother worth (Matricaria chamomilla) extract on Ichthyophtirius multifilus parasite treatment in sail fin molly (Poecilia latipinna) ornamental Fish. APCBEE Procedia. 2012; 4: 6-11.
  29. Ling F., Wang J.G., Lu, C., Wang G.X., Lui Y.H., Gong X.N. Effects of aqueous extracts of Capsicum frutescens (Solanaceae) against of the fish ectoparasite Ichthyophthirius multifiliis. Parasitol. Res. 2012; 111: 841-848.
  30. Yi Y. L., Lu C., Hu X. G., Ling F., Wang G. X. Antiprotozoal activity of medicinal plants against Ichthyophthirius multifiliis in goldfish (Carassius auratus). Parasitology Research. 2012; 111: 1771-1778.
  31. Ling F., Lu C., Tu X., Yi Y., Huang A., Zhan Q., Wang G. Antiprotozoal screening of traditional medicinal plants: Evaluation of crude extract of Psoralea corylifolia against Ichthyophthirius multifiliis in goldfish. Parasitology Research. 2013; 112: 2331-2340.
  32. Fu Y.W., Zhang Q.Z., Xu D.H., Xia H., Cai X.X., Wang B., et al. Parasiticidal effects of Morus alba root bark extracts against Ichthyophthirius multifiliis infecting grass carp. Dis. Aquat. Organ. 2014a; 108, 129-136.
  33. Yao J.Y., Zhou Z.M., Li X.L., Yin W.L., Ru H.S., Pan X.Y., et al. Antiparasitic efficacy of dihydrosanguinarine and dihydrochelerythrine from Macleaya microcarpa against Ichthyophthirius multifiliis in richadsin (Squaliobarbus curriculus). Veterinary Parasitology. 2011; 183: 8-13.
  34. Zhang Q.Z., Xu D.H., Klesius P.H. Evaluation of an antiparasitic compound extracted from Galla chinensis against fish parasite Ichthyophthirius multifiliis. Veterinary Parasitology. 2013; 198(1-2): 45-53.
  35. Shan X.F., Kang Y.H., Bian Y., Gao Y.h., Wang W.L., Qian A.D. Isolation of active compounds from methanol extracts of Toddalia asiatica against Ichthyophthirius multifiliis in gold fish (Carassius auratus). Veterinary Parasitology. 2014; 199(3-4): 250-254. https://doi.org/10.1016/j.vetpar.2013.10.021
  36. Fu Y.M., Zhang Q.Z., Xu D.H., Jiang J.H., Wang B. Antiparasitic effect of cynatratoside-C from Cynanchum atratum against Ichthyophthirius multifiliis on grass carp. J Agric Food Chem. 2014b; 62(29): 7183-7189. https://doi.org/10.1021/jf5018675
  37. Fu Y.W., Wang B., Zhang Q.Z., Xu D.H., Liu Y.M., Hou T.L., Guo S.Q. Efficacy and antiparasitic mechanism of 10-gingerol isolated from ginger Zingiber officinale against Ichthyophthirius multifiliis in grass carp. Veterinary Parasitology. 2019; 265: 74-84.
  38. Alavinia S.J., Mirzargar S.S., Rahmati‐Holasoo H., Mousavi H. E. The in vitro and in vivo effect of tannic acid on Ichthyophthirius multifiliis in zebrafish (Danio rerio) to treat ichthyophthiriasis. Journal of Fish Diseases. 2018: 41(12), 1793-1802.
  39. Alijanpour Z., Rahmati-Holasoo H., Ebrahimzadeh Mousavi H., Saeed Mirzargar S., Sharifzadeh A., Nasiri A. In vitro study of effects of alcoholic extract of Chelidonium majus L. on Ichthyophthirius multifiliis theronts. J. Fisheries. 2022; 75(3), 405-417.
  40. Rahmati-Holasoo H., Moosavi, M. S. J., Mousavi, H. E., Mirzargar, S. S., & Mirghaed, A. T. Effect of ethanol extract of Zataria multiflora on Ichthyophthirius multifiliis tomonts and theronts in Danio rerio. Journal of Veterinary Research. 2021; 76(2): 205-214.
  41. Yazdani Anaraki E., Mirzargar S.S., Rahmati Holasoo H., Sharifzade A., Ebrahimzade Musavi H. A. In vitro study of short-term antiparasitic effect of alcoholic extract of Terminalia catappa L. leaves on Ichthyophthirius multifiliis theronts. Iranian Journal of Fisheries Sciences. 2021; 20(4): 1138-1148.
  42. Bayati R., Asadi-Gharneh H.A. Study of Biochemical Compounds from Extract of Peel, Seed and Fruit Juice of some Pomegranate Cultivars (Punica granatum L.). J. Medicinal plants and By-Products. 2019; 8(2): .133-141.
  43. Lansky E, Shubert S, Neeman I. Pharmacological and therapeutic of pomegranate. Martínez-Tomé J, ed Production, processing and marketing of pomegranate in the Mediterranean region: Advances in research and technology Zaragoza: CIHEAM, 1997; 5: 231-235.
  44. Guivara J.M. The in vitro action of plants on Vibrio cholera. Revista in Gastroenterologia del Peru. 1994; 14: 27-31
  45. Afaq F., Saleem M., Krueger C.G., Reed J.D., Mukhtar H. Anthocyanin- and Hydrolyzable Tannin-rich Pomegranate Fruit Extract Modulates MAPK and NFkappaB Pathways and Inhibits Skin Tumorigenesis in CD-1 mice. International Journal of Cancer. 2005; 113: 423 -433. https://doi.org/10.1002/ijc.20587
  46. Prashanth D.J., Asha M.K., Amit A. Antibacterial Activity of Punica granatum. Fitoterapia 2001; 72: 171-173.
  47. Dutta B.K., Rahman I., Das TK. Antifungal Activity of Indian Plant Extracts. Mycoses 1998; 41: 535-536. https://doi.org/10.1111/j.1439-0507.1998.tb00718.x
  48. Gharzouli K., Khennouf S., Amira S., Gharzouli A. Effects of aqueous extracts from Quercus ilex l. root Bark, Punica granatum L. fruit peel and Artemisia Herba-alba Asso leaves on ethanol-induced gastric damage in rats. Phytotheraphy Research 1999; 13: 42-45. https://doi.org/10.1002/(SICI)1099-1573(199902)13:1<42::AID-PTR383>3.0.CO;2-2
  49. Aviram M., Dornfeld L., Rosenblat M., Volkova N., Kaplan M., Coleman R., Hayek T., Presser D., Fuhrman B. Pomegranate Juice Consumption Reduces Oxidative Stress, Atherogenic Modifications to LDL and Platelet Aggregation: Studies in Humans and in Atherosclerotic Apolipoprotein E-deficient mice. The American J. Clinical Nutrition 2000; 71: 1062-1076.
  50. Lee S.I., Kim B.S., Kim K.S., Lee S., Shin K.S., Lim J.S. Immune-suppressive Activity of Punicalagin via Inhibition of NFAT Activation. Biochemical and Biophysical Research Communications 2008; 11: 799-803. https://doi.org/10.1016/j.bbrc.2008.04.150
  51. Thresiamma K.C., Kuttan R. Inhibition of Liver Fibrosis by Ellagic Acid. Indian J. Physiology and Pharmacology 1996; 40: 363-366.
  52. Rosenblat M., Draganov D., Watson C.E., Bisgaier C.L., La Du B.N., Aviram M. Mouse macrophage Paraoxonase 2 activity is increased whereas cellular Paraoxonase 3 activity is decreased under oxidative stress. Arteriosclerosis, Thrombosis and Vascular Biology. 2003; 23(3): 468-474.
  53. Finney D.J. Statistical logic in the monitoring of reactions to therapeutic drugs. Methods of Information in Medicine. 1971a; 10(04), 237-245.
  54. Finney D.J. Statistical aspects of monitoring for dangers in drug therapy. Methods of Information in Medicine. 1971b; 10(01), 1-8.
  55. Liang J.H., Fu Y.W., Zhang Q.Z., Xu D.H., Wang B., Lin D.J. Identification of effect of two flavonoids from root bark of Morus alba against Ichthyophthirius multifiliis in grass carp. J. Agric. Food Chem. 2015; 63: 1452-1459.
  56. Shinn A.P., Picón-Camacho S.M., Bron J.E., Conway D., Yoon G.H., Guo F.C., Taylor NG. The anti-protozoal activity of bronopol on the key life-stages of Ichthyophthirius multifiliis Fouquet, 1876 (Ciliophora). Veterinary Parasitology. 2012; 25: 186(3-4):229-36.
  57. Picón-Camacho S.M., Taylor N.G., Bron J.E., Guo F.C., Shinn A.P. Effects of long duration, low dose bronopol exposure on the control of Ichthyophthirius multifiliis (Ciliophora), parasitising rainbow trout (Oncorhynchus mykiss Walbaum). Veterinary Parasitology. 2012; 186(3-4): 237-244.
  58. Martinez-Ortiz-de-Montellano C., Vargas-Maganad J.J., canul-Ku H.L., Miranda-Soberanis R., Capetillo-Leal C., Sandoval-Castro C.A., Hoste, H., Torres-Acosta J. F. J. Effect of a tropical tannin-rich plant Lysiloma latisiliquum on adult populations of Haemonchus contortus in sheep. Veterinary Parasitology. 2010; 172(3-4): 283-290. https://doi.org/10.1016/j.vetpar.2010.04.040
  59. Mathiessen H., Jaafar R.M. Al‐Jubury A., von Gersdorff Jørgensen L., Kania P.W., Buchmann K. Comparative in vitro and in vivo effects of feed additives on rainbow trout response to Ichthyophthirius multifiliis. North American J. Aquaculture. 2021; 83(2): 67-77.
  60. Alavinia S.J., Mirzargar S.S., Rahmati-Holasoo H., Mousavi H. In vitro investigation of short-term antiparasitic effect of tannic acid on Ichthyophthirius multifiliis theronts. J. Veterinary Research. 2019; 74(2): 219-227.
  61. Rahmati Taghjeh Hassan J., Yakhchali M., Nekoueifard A. Study on effects of plants extracts of Thymus vulgaris and Lavandula angustifolia on Ichthyophthirius multifiliis in rainbow trout (Oncorhynchus mykiss). Iranian J. Animal Science. 2024; Available Online.