Environmental Science and Pollution Research

, Volume 26, Issue 9, pp 8798–8807 | Cite as

Immunostimulating effects of Ginkgo biloba extract against toxicity induced by organophosphate pesticide, diazinon in rainbow trout, Oncorhynchus mykiss: innate immunity components and immune-related genes

  • Saeed HajirezaeeEmail author
  • Ahmad Rafieepour
  • Shafigh Shafiei
  • Ruhollah Rahimi
Research Article


The immunostimulating and therapeutic properties of Ginkgo biloba (GB) have always been the focus of traditional medicine over thousands of years. During last decade, special attentions were paid to use of GB in aquaculture to enhance fish health and survival. In the present study, we investigated for the first time the immunogenic effects of dietary GB against oxidative and toxicity induced by organophosphate pesticide, diazinon. In non-diazinon-exposed fish, the plasma total immunoglobulin, lysozyme activity, and peroxidase activity significantly elevated after 60-day experiment in fish supplemented with 1 and 2 g GB/kg diet (p < 0.05). The respiratory burst activity and complement activity significantly increased only in groups supplemented with 0.5 g GB/kg diet (p < 0.05). Furthermore, the peroxidase activity, total immunoglobulin, and lysozyme activity significantly declined in groups supplemented with 4 g GB/kg diet during feeding trial (p < 0.05). There were no significant differences in expression of interleukin 1 beta (IL-1β) and transforming growth factor beta 1 (TGF-β1) genes in kidney between control group (non-GB-supplemented fish) and GB-supplemented fish (p > 0.05). In diazinon-exposed fish, all immunity components significantly decreased during exposure in control and those fed 0.5 and 4 g GB/kg diet (p < 0.05). In fish fed 1 and 2 g GB/kg diet, no alternations were found in immunity components during exposure period (p > 0.05). In addition, diazinon induced the expression of IL-1β and TGF-β1 genes in control and fish fed 0.5 and 4 g GB/kg diet (p < 0.05). No significant changes were observed in expression of IL-1β and TGF-β1 genes in fish supplemented with 1 and 2 g GB/kg (p > 0.05). In conclusion, the results of the present study suggest an immunogenic role for dietary GB at optimum dietary levels (1–2 g GB/kg diet) against toxicity induced by diazinon. Nevertheless, GB at high dietary levels (4 g GB/kg diet) showed immunosuppressive effects, which makes it necessary to optimize its levels in diet.


Fish Pesticide Plant extract Immunity Gene expression 



The authors express their sincere gratitude to the people who lent their time, advice, and support to this study.

Funding information

This study was supported by university of Jiroft under the grant No. 4813-97-7.


  1. Ahmadi K, Mirvaghefei AR, Banaee M, Vosoghei AR (2014) Effects of long-term diazinon exposure on some immunological and haematological parameters in rainbow trout Oncorhynchus mykiss (Walbaum, 1792). Toxicol Environ Heal Sci 6(1):1–7Google Scholar
  2. Andreson DP, Zeeman MG (1995) Immunotoxicology in fish. In: Rand GM (ed) Fundamentals of aquatic toxicology. Taylorand Francis, Washington, pp 371–404Google Scholar
  3. Arufe MI, Arellano JM, García L, Albendín G, Sarasquete C (2007) Cholinesterase activity in gilthead seabream (Sparus aurata) larvae: characterization and sensitivity to the organophosphate azinphosmethyl. Aquat Toxicol 84(3):328–336Google Scholar
  4. Awad E, Mitchell WJ, Austin B (2011) Effect of dietary supplements on cytokine gene expression in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 34(8):629–634Google Scholar
  5. Banaee M, Mirvagefei AR, Rafei GR, Majazi Amiri B (2008) Effect of sub-lethal diazinon concentrations on blood plasma biochemistry. Int J Environ Res 2(2):189–198Google Scholar
  6. Banaee M, Sureda A, Mirvaghefi AR, Ahmadi K (2011) Effects of diazinon on biochemical parameters of blood in rainbow trout (Oncorhynchus mykiss). Pestic Biochem Physiol 99(1):1–6Google Scholar
  7. Banaee M, Sureda A, Shahaf S, Fazilat N (2015) Protective effects of silymarin extract on malthion-induced zebra cichlid (Cichlasomanigrofasciatum) hepatotoxicity. Iran J Toxicol 9(28):1239–1246Google Scholar
  8. Bridi R, Crossetti FP, Steffen VM, Henriques AT (2001) The antioxidant activity of standardized extract of Ginkgo biloba (EGb 761) in rats. Phytother Res 15(5):449–451Google Scholar
  9. Cao F, Lu Y, Zhang X, Zhao L, Yang J, Qin X, Yu W (2015) Effects of dietary supplementation with fermented Ginkgo leaves on innate immunity, antioxidant capability, lipid metabolism, and disease resistance against Aeromonas hydrophila infection in blunt snout bream (Megalobrama amblycephala). Isr J Aquac 67:1202–1212Google Scholar
  10. Çavușoğlu K, Yapar K, Yalcin E (2009) Antioxidant potential of Ginkgo biloba leaf extract against uranium-induced genotoxicity and oxidative stress in albino mice. Fresenius Environ Bull 18(9):1551–1558Google Scholar
  11. Çavuşoğlu K, Yapar K, Oruç E, Yalçın E (2011) Protective effect of Ginkgo biloba L. leaf extract against glyphosate toxicity in Swiss albino mice. J Med Food 14(10):1263–1272Google Scholar
  12. Chakraborty SB, Hancz C (2011) Application of phytochemicals as immunostimulant, antipathogenic and antistress agents in finfish culture. Rev Aquac 3(3):103–119Google Scholar
  13. Chan PC, Xia Q, Fu PP (2007) Ginkgo biloba leave extract: biological, medicinal, and toxicological effects. J Environ Sci Health Part C 25(3):211–244Google Scholar
  14. Chávez-Morales RM, Jaramillo-Juárez F, Posadas del Río FA, Reyes-Romero MA, Rodríguez-Vázquez ML, Martínez-Saldaña MC (2011) Protective effect of Ginkgo biloba extract on liver damage by a single dose of CCl 4 in male rats. Hum Exp Toxicol 30(3):209–216Google Scholar
  15. Citarasu T, Jayarani TV, Babu MM, Marian MP (1999) Use of herbal bio-medicinal products in aquaculture of shrimp. In Aqua-Terr Annual Symposium, School of Biological Sciences, MK University, Madurai (p. 83)Google Scholar
  16. Citarasu T, Babu MM, Punitha SMJ, Ramalingam V, Marian MP (2001) Control of pathogenic bacteria using herbal biomedical products in the larviculture system of Peneaus monodon. In: Int. Con. Ad. Tech. Fish Mar. Sci. Indian Council of Agricultural Science, M.S. University, Nagercoil, IndiaGoogle Scholar
  17. Citarasu T, VenketRamalingam K, Raja JeyaSekar R, MichealBabu M, Marian MP (2002) Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoraleacorylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae. Aquac Int 11:583–595Google Scholar
  18. Cooper CM (1993) Biological effects of agriculturally derived surface water pollutants on aquatic systems—a review. J Environ Qual 22(3):402–408Google Scholar
  19. Demers NE, Bayne CJ (1997) The immediate effects of stress on hormones and plasma lysozyme in rainbow trout. Dev Comp Immunol 21(4):363–373Google Scholar
  20. Dunier M, Siwicki AK (1993) Effects of pesticides and other organic pollutants in the aquatic environment on immunity of fish: a review. Fish Shellfish Immunol 3(6):423–438Google Scholar
  21. Eder KJ, Leutenegger CM, Wilson BW, Werner I (2004) Molecular and cellular biomarker responses to pesticide exposure in juvenile Chinook salmon (Oncorhynchus tshawytscha). Mar Environ Res 58(2–5):809–813Google Scholar
  22. Eder KJ, Clifford MA, Hedrick RP, Köhler HR, Werner I (2008) Expression of immune-regulatory genes in juvenile Chinook salmon following exposure to pesticides and infectious hematopoietic necrosis virus (IHNV). Fish Shellfish Immunol 25(5):508–516Google Scholar
  23. El-Gendy KS, Aly NM, El-Sebae AH (1998) Effects of edifenphos and glyphosate on the immune response and protein biosynthesis of bolti fish (Tilapia nilotica). J Environ Sci Health Part B 33(2):135–149Google Scholar
  24. Fatima M, Mandiki SNM, Douxfils J, Silvestre F, Coppe P, Kestemont P (2007) Combined effects of herbicides on biomarkers reflecting immune–endocrine interactions in goldfish: immune and antioxidant effects. Aquat Toxicol 81(2):159–167Google Scholar
  25. Galina J, Yin G, Ardo L, Jeney Z (2009) The use of immunostimulating herbs in fish. An overview of research. Fish Physiol Biochem 35(4):669–676Google Scholar
  26. Galloway T, Handy R (2003) Immunotoxicity of organophosphorous pesticides. Ecotoxicol 12(1–4):345–363Google Scholar
  27. Girón-Pérez MI, Santerre A, Gonzalez-Jaime F, Casas-Solis J, Hernández-Coronado M, Peregrina-Sandoval J, Takemura A, Zaitseva G (2007) Immunotoxicity and hepatic function evaluation in Nile tilapia (Oreochromis niloticus) exposed to diazinon. Fish Shellfish Immunol 23(4):760–769Google Scholar
  28. Guilherme S, Gaivão I, Santos MA, Pacheco M (2012) DNA damage in fish (Anguilla anguilla) exposed to a glyphosate-based herbicide–elucidation of organ-specificity and the role of oxidative stress. Mutat Res Genet Toxicol Environ Mutagen 743(1):1–9Google Scholar
  29. Kampkötter A, Pielarski T, Rohrig R, Timpel C, Chovolou Y, Wätjen W, Kahl R (2007) The Ginkgo biloba extract EGb761 reduces stress sensitivity, ROS accumulation and expression of catalase and glutathione S-transferase 4 in Caenorhabditiselegans. Pharmacol Res 55(2):139–147Google Scholar
  30. Khoshbavar-Rostami HA, Soltani M, Hassan HMD (2006) Immune response of great sturgeon (Huso huso) subjected to long-term exposure to sublethal concentration of the organophosphate, diazinon. Aquac 256(1–4):88–94Google Scholar
  31. Kleijnen J, Knipschild P (1992) Ginkgo biloba. Lancet 340(8828):1136–1139Google Scholar
  32. Kreutz LC, Barcellos LJG, de Faria VS, de Oliveira ST, Anziliero D, dos Santos ED, Pivato M, Zanatta R (2011) Altered hematological and immunological parameters in silver catfish (Rhamdia quelen) following short term exposure to sublethal concentration of glyphosate. Fish Shellfish Immunol 30(1):51–57Google Scholar
  33. Krithika R, Verma RJ (2014) Ameliorative effects of phyllanthin on carbon tetrachloride-induced hepatic oxidative damage in mice. Asian Pac J Trop Dis 4:S64–S70Google Scholar
  34. Lackner R (1998) “Oxidative stress” in fish by environmental pollutants. In: Fish ecotoxicology. Birkhäuser, Basel, pp 203–224Google Scholar
  35. Li X, Liu L, Zhang Y, Fang Q, Li Y, Li Y (2013) Toxic effects of chlorpyrifos on lysozyme activities, the contents of complement C3 and IgM, and IgM and complement C3 expressions in common carp (Cyprinus carpio L.). Chemosphere 93(2):428–433Google Scholar
  36. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25(4):402–408Google Scholar
  37. Loganathan BG, Kannan K (1991) Time perspectives of organochlorine contamination in the global. Mar Pollut Bull 22(12):582–584Google Scholar
  38. Loganathan BG, Kannan K (1994) Global organochlorine contamination trends: an overview. Ambio 23:187–191Google Scholar
  39. Ma J, Feng Y, Xie W, Yang L, Li X (2015) Immune response in common carp (Cyprinus carpio L.) following glyphosate-exposure. Toxin Rev 34(4):184–189Google Scholar
  40. Marcocci L, Maguire JJ, Droylefaix MT, Packer L (1994a) The nitric oxide-scavenging properties of Ginkgo biloba extract EGb 761. Biochem Biophys Res Commun 201(2):748–755Google Scholar
  41. Marcocci L, Packer L, Droy-Lefaix MT, Sekaki A, Gardès-Albert M (1994b) [46] antioxidant action of Ginkgo biloba extract EGb 761. In Methods in enzymology (Vol. 234, pp. 462-475). Academic PressGoogle Scholar
  42. Miandare HK, Farahmand H, Akbarzadeh A, Ramezanpour S, Kaiya H, Miyazato M, Rytkönen KT, Nikinmaa M (2013) Developmental transcription of genes putatively associated with growth in two sturgeon species of different growth rate. Gen Comp Endocrinol 182:41–47Google Scholar
  43. Mondon JA, Duda S, Nowak BF (2000) Immune response of greenback flounder Rhombosoleatapirina after exposure to contaminated marine sediment and diet. Mar Environ Res 50(1–5):443–450Google Scholar
  44. Mora S, Diaz-Veliz G, Lungenstrass H, Garcia-Gonzalez M, Coto-Morales T, Poletti C, De Lima TCM, Herrera-Ruiz M, Tortoriello J (2005) Central nervous system activity of the hydroalcoholic extract of Casimiroaedulis in rats and mice. J Ethnopharmacol 97(2):191–197Google Scholar
  45. Nayak AS, Lage CR, Kim CH (2007) Effects of low concentrations of arsenic on the innate immune system of the zebrafish (Danio rerio). Toxicol Sci 98(1):118–124Google Scholar
  46. Nowell LH, Capel PD, Dileanis PD (1999) Pesticides in stream sediment and aquatic biota: distribution, trends, and governing factors. CRC Press, Boca RatonGoogle Scholar
  47. Nya EJ, Austin B (2009) Use of dietary ginger, Zingiber officinale roscoe, as an immunostimulant to control Aeromonas hydrophila infections in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis 32(11):971–977Google Scholar
  48. Ortuno J, Esteban MA, Meseguer J (2001) Effects of short-term crowding stress on the gilthead seabream (Sparusaurata L.) innate immune response. Fish Shellfish Immunol 11(2):187–197Google Scholar
  49. Parvez S, Raisuddin S (2005) Protein carbonyls: novel biomarkers of exposure to oxidative stress-inducing pesticides in freshwater fish Channapunctata (Bloch). Environ Toxicol Pharmacol 20(1):112–117Google Scholar
  50. Prasad S, Variyur Padhyoy KB (1993) Chemical investigation of some commonly used spices. Aryavaidyan 6(4):262–267Google Scholar
  51. Puebla-Pérez AM, Lozoya X, Villaseñor-García MM (2003) Effect of Ginkgo biloba extract, EGb 761, on the cellular immune response in a hypothalamic–pituitary–adrenal axis activation model in the rat. Int Immunopharmacol 3(1):75–80Google Scholar
  52. Qichun H, Xintian Z, Xiaoyan Y (2012) The modulatory role of Ginkgo biloba extract on improving the immune function of weaned piglets. J Northwest Agri For Univ 11:23–28Google Scholar
  53. Quade MJ, Roth JA (1997) A rapid, direct assay to measure degranulation of bovine neutrophil primary granules. Vet Immunol Immunopathol 58(3–4):239–248Google Scholar
  54. Rabie M, Asri Y, Ahmadi K (2016) Effect of Milk thistle plant, Vitis vinifera extract on immune system of rainbow trout (Oncorhynchus mykiss) challenge by diazinon. Int J Aqua Biol 4(3):208Google Scholar
  55. Raj S, Gothandam KM (2014) Hepatoprotective effect of polyphenols rich methanolic extract of Amorphophalluscommutatus var. wayanadensis against CCl4 induced hepatic injury in swiss albino mice. Food Chem Toxicol 67:105–112Google Scholar
  56. Raymond M (1985) Log-probit analysis program presentation for a microcomputer. Entomologie Médicale et Parasitologie 23(2):117–121Google Scholar
  57. Reverter M, Bontemps N, Lecchini D, Banaigs B, Sasal P (2014) Use of plant extracts in fish aquaculture as an alternative to chemotherapy: current status and future perspectives. Aquac 433:50–61Google Scholar
  58. Secombes CJ, Bird S, Cunningham C, Zou J (1999) Interleukin-1 in fish. Fish Shellfish Immunol 9(4):335–343Google Scholar
  59. Shaaban AA, Shaker ME, Zalata KR, El-Kashef HA, Ibrahim TM (2014) Modulation of carbon tetrachloride-induced hepatic oxidative stress, injury and fibrosis by olmesartan and omega-3. Chem Biol Interact 207:81–91Google Scholar
  60. Shelley LK, Balfry SK, Ross PS, Kennedy CJ (2009) Immunotoxicological effects of a sub-chronic exposure to selected current-use pesticides in rainbow trout (Oncorhynchus mykiss). Aquat Toxicol 92(2):95–103Google Scholar
  61. Sivaram JJ, Wu Z (2004) Effect of Chinese herbal medicine on non-specific immunity of Jian common carp (Cyprinuscarpio var. Jian). J Dalian Fish Univ 17:114–119Google Scholar
  62. Siwicki AK, Anderson DP (1993) Nonspecific defense mechanisms assay in fish: II. Potential killing activity of neutrophils and macrophages, lysozyme activity in serum and organs and total immunoglobulin level in serum. Fish Disease Diagnosis and Prevention Methods, FAO-Project GCP/INT/526/JPN, IFI Olsztyn, pp 105–112Google Scholar
  63. Siwicki AK, Terech-Majewska E, Grudniewska J, Malaczewska J, Kazun K, Lepa A (2010) Influence of deltamethrin on nonspecific cellular and humoral defense mechanisms in rainbow trout (Oncorhynchus mykiss). Environ Toxicol Chem 29(3):489–491Google Scholar
  64. Slaninova A, Smutna M, Modra H, Svobodova Z (2009) REVIEWS oxidative stress in fish induced by pesticides. Neuro Endocrinol Lett 30(1):2Google Scholar
  65. Sun H (2009) The effects of Ginkgo biloba leaf extract (GBE) on cytochrome P4503A and lysozyme activity in crucian carp (Carassius auratus). Fish Sci (Dalian) 28(12):760–762Google Scholar
  66. Tan X, Sun Z, Liu Q, Ye H, Zou C, Ye C, Lin H (2018) Effects of dietary ginkgo biloba leaf extract on growth performance, plasma biochemical parameters, fish composition, immune responses, liver histology, and immune and apoptosis-related genes expression of hybrid grouper (Epinephelus lanceolatus♂× Epinephelus fuscoguttatus♀) fed high lipid diets. Fish Shellfish Immunol 72:399–409Google Scholar
  67. Tao R, Wang CZ, Ye JZ, Zhou H, Chen HX, Zhang YS (2014) Antibacterial/antifungal activity and synergistic interactions between C70–C120 polyprenol homologs from Ginkgo biloba L. leaves and the corresponding synthetic derivatives. Eur Food Res Technol 239(4):587–594Google Scholar
  68. Tellez-Bañuelos MC, Santerre A, Casas-Solis J, Bravo-Cuellar A, Zaitseva G (2009) Oxidative stress in macrophages from spleen of Nile tilapia (Oreochromis niloticus) exposed to sublethal concentration of endosulfan. Fish Shellfish Immunol 27(2):105–111Google Scholar
  69. Tian YM, Tian HJ, Zhang GY, Dai YR (2003) Effects of Ginkgo biloba extract (EGb 761) on hydroxyl radical-induced thymocyte apoptosis and on age-related thymic atrophy and peripheral immune dysfunctions in mice. Mech Ageing Dev 124(8–9):977–983Google Scholar
  70. Tsuda T, Kojima M, Harada H, Nakajima A, Aoki S (1997) Acute toxicity, accumulation and excretion of organophosphorous insecticides and their oxidation products in killifish. Chemosphere 35(5):939–949Google Scholar
  71. Tukmechi A, Rezaee J, Nejati V, Sheikhzadeh N (2014) Effect of acute and chronic toxicity of paraquat on immune system and growth performance in rainbow trout, Oncorhynchus mykiss. Aquac Res 45(11):1737–1743Google Scholar
  72. Valavanidis A, Vlahogianni T, Dassenakis M, Scoullos M (2006) Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants. Ecotoxicol Environ Saf 64(2):178–189Google Scholar
  73. Van Hai N (2015) The use of medicinal plants as immunostimulants in aquaculture: a review. Aquac 446:88–96Google Scholar
  74. Velisek J, Svobodova Z, Machova J (2009) Effects of bifenthrin on some haematological, biochemical and histopathological parameters of common carp (Cyprinus carpio L.). Fish Physiol Biochem 35(4):583–590Google Scholar
  75. Villaseñor-García MM, Lozoya X, Osuna-Torres L, Viveros-Paredes JM, Sandoval-Ramírez L, Puebla-Pérez AM (2004) Effect of Ginkgo biloba extract EGb 761 on the nonspecific and humoral immune responses in a hypothalamic–pituitary–adrenal axis activation model. Int Immunopharmacol 4(9):1217–1222Google Scholar
  76. Wang D, Zhao Y, Sun Y, Yang X (2014) Protective effects of Ziyang tea polysaccharides on CCl4-induced oxidative liver damage in mice. Food Chem 143:371–378Google Scholar
  77. Weeks BA, Warinner JE (1984) Effects of toxic chemicals on macrophage phagocytosis in two estuarine fishes. Mar Environ Res 14(1–4):327–335Google Scholar
  78. Yang XY, Lin YX, Li Y (2008) Effect of ginkgo biloba extract on growth performance, slaughter performance and immune index in broilers [J]. J Fujian Agri For Univ (Natural Science Edition) 3:016Google Scholar
  79. Yonar SM (2013) Toxic effects of malathion in carp, Cyprinuscarpiocarpio: protective role of lycopene. Ecotoxicol Environ Saf 97:223–229Google Scholar
  80. Zahran E, Risha E, Awadin W, Palić D (2018) Acute exposure to chlorpyrifos induces reversible changes in health parameters of Nile tilapia (Oreochromis niloticus). Aquat Toxicol 197:47–59Google Scholar
  81. Zhang Z, Chen S, Mei H, Xuan J, Guo X, Couch L, Dobrovolsky VN, Guo L, Mei N (2015) Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells. Sci Rep 5:14633Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Saeed Hajirezaee
    • 1
    Email author
  • Ahmad Rafieepour
    • 1
  • Shafigh Shafiei
    • 2
  • Ruhollah Rahimi
    • 3
  1. 1.Department of Fisheries and Environmental Sciences, Faculty of Natural ResourcesUniversity of JiroftJiroftIran
  2. 2.Department of food hygiene and quality control, Faculty of veterinary medicineShahrekord UniversityShahrekordIran
  3. 3.Department of Fisheries and Environmental Sciences, Faculty of Natural Resources and Earth SciencesShahrekord UniversityShahrekordIran

Personalised recommendations