Dietary supplementation of drumstick tree, Moringa oleifera, improves mucosal immune response in skin and gills of seabream, Sparus aurata, and attenuates the effect of hydrogen peroxide exposure

  • Abdallah Tageldein MansourEmail author
  • Cristóbal Espinosa
  • Jose María García-Beltrán
  • Liang Miao
  • Diana C. Ceballos Francisco
  • Ahmed Saud Alsaqufi
  • M. Ángeles Esteban


In aquatic animals, the mucosal barrier is the first line of innate immune defence against external chemicals and pathogens. In this study, the effects of dietary Moringa oleifera leaf (MOL) supplementation on skin and gill mucosal immunity, antioxidants and stress responses were evaluated in seabream (Sparus aurata) fingerlings exposed to hydrogen peroxide (H2O2). A total of 144 specimens (10.11 ± 0.41 g) were divided into four treatments (three replicates per treatment contained 12 specimens each) and fed a non-supplemented control diet or a 1, 2.5 or 5% MOL-supplemented diet. After three weeks of feeding, six specimens from each aquarium were sampled for blood, mucus and tissues. The other six fish in each aquarium were subjected to H2O2 exposure. The results revealed that MOL did not negatively affect either cortisol or glucose levels. MOL supplementation significantly (P < 0.05) improved skin mucosal immunity-related characteristics, including phosphatase, peroxidase and lysozyme activity and IgM levels. Additionally, MOL upregulated the expression of antioxidant genes (sod and cat), an anti-inflammatory gene (tgf-β), tight junction protein genes (occludin and zo-1), c3, and igm in both the skin and gills. However, H2O2 exposure significantly (P < 0.05) increased both cortisol and glucose levels and disrupted skin mucosal immune function by significantly (P < 0.05) decreasing phosphatase, peroxidase, protease, antiprotease and lysozyme activity and IgM levels. H2O2 exposure severely decreased the mRNA levels of the studied genes. MOL dietary supplementation at the 5% level successfully attenuated the negative effects of H2O2 on the mucosal immune response in both the skin and gills. In conclusion, dietary MOL supplementation at the 5% level is recommended to improve S. aurata mucosal immune function under both normal and stress conditions. Additionally, exposure to H2O2 disrupts the mucosal immunity of fish. This contributes knowledge on the routes involved in mucosal innate immunity and could help to understand the fish resistance against chemicals exposure.

Graphical abstract


Phytochemical Mucosal immunity H2O2 Stress Seabream 



The authors wish to thank Alexandria University, Egypt, for the post-doctoral grant (Alex-GYR) for Dr. Abdallah T. Mansour.

Compliance with ethical standards

Competing interests

The authors declare that they have no conflict of interest.


  1. Abdel-Latif M, Sakran T, Badawi YK, Abdel-Hady DS (2018) Influence of Moringa oleifera extract, vitamin C, and sodium bicarbonate on heat stress-induced HSP70 expression and cellular immune response in rabbits. Cell Stress Chaperones 23:975–984. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ahmed MK, Islam KN, Ibrahim M, Sultana GNN, Khan MS, Akter MS, Shahinuzzaman A, Kundu GK, Hossain A (2016) Oxidative stress mediated antioxidant enzyme responses in tilapia (Oreochromis mossambicus) and silver carp (Hypophthalmichthys molitrix) fingerlings during hypoxic transportation and reoxygenation. Biores Commun 2:264–269Google Scholar
  3. Anwar F, Latif S, Ashraf M, Gilani A (2007) Moringa oleifera: a food plant with multiple medicinal uses. Phytother Res 21:17–25. CrossRefPubMedGoogle Scholar
  4. Arndt RE, Wagner EJ (1997) The toxicity of hydrogen peroxide to rainbow trout, Oncorhynchus mykiss, and cutthroat trout, Oncorhynchus clarki, fry and fingerlings. J World Aquacult Soc 28:150–157. CrossRefGoogle Scholar
  5. Arndt RE, Wagner EJ, Routledge MD (2001) Reducing or withholding hydrogen peroxide treatment during a critical stage of rainbow trout development: effects on eyed eggs, hatch, deformities, and fungal control. N Am J Aquac 63:161–166.<0161:ROWHPT>2.0.CO;2 CrossRefGoogle Scholar
  6. Avendaño-Herrera R, Magariños B, Irgang R, Toranzo AE (2006) Use of hydrogen peroxide against the fish pathogen Tenacibaculum maritimum and its effect on infected turbot (Scophthalmus maximus). Aquaculture 257:104–110. CrossRefGoogle Scholar
  7. Awad E, Awaad A (2017) Role of medicinal plants on growth performance and immune status in fish. Fish Shellfish Immunol 67:40–54. CrossRefPubMedGoogle Scholar
  8. Beaulieu AK, Greenlees K, Haley C, Kahn L, Oeller M, Bell T, Oriani JA (1992) Aquaculture drug use: answers to commonly asked questions.FDA workshop requirements for investigational new animal drugs—eastern fish health group and the American fisheries society fish health section. U.S. Food and Drug Administration, Center for Veterinary Medicine, RockvilleGoogle Scholar
  9. Bols NC, Brubacher JL, Ganassin RC, Lee LE (2001) Ecotoxicology and innate immunity in fish. Dev Comp Immunol 25:853–873. CrossRefPubMedGoogle Scholar
  10. Bowers J, Speare DJ, Burka JF (2002) The effects of hydrogen peroxide on the stress response of Atlantic Salmon (Salmo salar). J Vet Pharmacol Ther 25:311–313. CrossRefPubMedGoogle Scholar
  11. Carrasco GA, van de Kar LD (2003) Neuroendocrine pharmacology of stress. Eur J Pharmacol 463:235–272. CrossRefPubMedGoogle Scholar
  12. Cuesta A, Meseguer J, Esteban M (2004) Total serum immunoglobulin M levels are affected by immunomodulators in seabream (Sparus aurata L.). Vet Immunol Immunopathol 101:203–210. CrossRefPubMedGoogle Scholar
  13. Duncan DB (1955) Multiple range and multiple F test. Biometric 11:1–42. CrossRefGoogle Scholar
  14. Elenkov I, Chrousos G (1999) Stress hormones, Th1/Th2 patterns, pro/antiinflammatory cytokines and susceptibility to disease. Trends Endocrinol Metab 10:359–368. CrossRefPubMedGoogle Scholar
  15. Elliott D (2000) Integumentary system. Academic Press, New YorkGoogle Scholar
  16. Esteban MA (2012) An overview of the immunological defenses in fish skin. ISRN Immunol:1–29. CrossRefGoogle Scholar
  17. Fayed WM, Khalil RH, Sallam GR, Mansour AT, Elkhayat BK, Omar EA (2019) Estimating the effective level of Yucca schidigera extract for improvement of the survival, haematological parameters, immunological responses and water quality of European seabass juveniles (Dicentrarchus labrax). Aquaculture Reports 15:100208. CrossRefGoogle Scholar
  18. Fernández-Alacid L, Sanahuja I, Ordóñez-Grande B, Sánchez-Nuño S, Viscor G, Gisbert E, Herrera M, Ibarz A (2018) Skin mucus metabolites in response to physiological challenges: a valuable non-invasive method to study teleost marine species. Sci Total Environ 644:1323–1335. CrossRefPubMedGoogle Scholar
  19. Fu M, Zou Z, Liu S, Lin P, Wang Y, Zhang Z (2012) Selenium-dependent glutathione peroxidase gene expression during gonad development and its response to LPS and H2O2 challenge in Scylla paramamosain. Fish Shellfish Immunol 33:532–542. CrossRefPubMedGoogle Scholar
  20. Furtado PS, Serra FP, Poersch LH, Wasielesky W (2014) Acute toxicity of hydrogen peroxide in juvenile white shrimp, Litopenaeus vannamei, reared in biofloc technology systems. Aquacult int 22:653–659. CrossRefGoogle Scholar
  21. Gbadamosi OK, Fasakin AE, Adebayo OT (2016) Hepatoprotective and stress - reducing effects of dietary Moringa oleifera extract against Aeromonas hydrophila infections and transportation-induced stress in Nile tilapia, Oreochromis niloticus (Linnaeus 1757) fingerlings. Int j environ agric res 2:121–128Google Scholar
  22. Gomez-Sanchez C, Milewich L, Holland OB (1977) Radioiodinated derivatives for steroid radioimmunoassay. Application to the radioimmunoassay of cortisol. J Lab Clin Med 89:902–909PubMedGoogle Scholar
  23. Guardiola FA, Cuesta A, Abellan E, Meseguer J, Esteban MA (2014) Comparative analysis of the humoral immunity of skin mucus from several marine teleost fish. Fish Shellfish Immunol 40:24–31. CrossRefPubMedGoogle Scholar
  24. Guardiola F, Dioguardi M, Parisi MG, Trapani MR, Meseguer J, Cuesta A, Cammarata M, Esteban MÁ (2015a) Evaluation of waterborne exposure to heavy metals in innate immune defences present on skin mucus of gilthead seabream (Sparus aurata). Fish shellfish Immunol 45:112–123. CrossRefPubMedGoogle Scholar
  25. Guardiola FA, Cuartero M, del Mar Collado-González M, Arizcún M, Díaz Baños FG, Meseguer J, Cuesta A, Esteban MA (2015b) Description and comparative study of physico-chemical parameters of the teleost fish skin mucus. Biorheology 52:247–256. CrossRefPubMedGoogle Scholar
  26. Guardiola F, Bahi A, Jiménez-Monreal A, Martínez-Tomé M, Murcia M, Esteban M (2018) Dietary administration effects of fenugreek seeds on skin mucosal antioxidant and immunity status of gilthead seabream (Sparus aurata L.). Fish Shellfish Immunol 75:357–364. CrossRefPubMedGoogle Scholar
  27. Gupta R, Kannana GM, Sharma M, Flora SJS (2005) Therapeutic effects of Moringa oleifera on arsenic-induced toxicity in rats. Environ Toxicol Pharmacol 20:456–464. CrossRefPubMedGoogle Scholar
  28. Hamed HS, El-Sayed YS (2019) Antioxidant activities of Moringa oleifera leaf extract against pendimethalin-induced oxidative stress and genotoxicity in Nile tilapia, Oreochromis niloticus (L.). Fish Physiol Biochem 45:71–82. CrossRefPubMedGoogle Scholar
  29. Hanif A, Bakopoulos V, Dimitriadis G (2004) Maternal transfer of humoral specific and non-specific immune parameters to sea bream (Sparus aurata) larvae. Fish Shellfish Immunol 17:411–435. CrossRefPubMedGoogle Scholar
  30. Hefni M (2013) Imunomodulator activity of aqueous extract of M. oleifera lam on immunity response of mice (Mus musculus) which infected with Salmonella typhi. Master thesis. University of Brawijaya. MalangGoogle Scholar
  31. Hisam EEA, Rofiee MS, Khalid AM, Jalaluddin AF, Yusof MIM, Idris MH, Ramli S, James RJ, Yoeng WJ, Kek TL, Salleh MZ (2018) Combined extract of Moringa oleifera and Centella asiatica modulates oxidative stress and senescence in hydrogen peroxide-induced human dermal fibroblasts. Turk J Biol 42:33–44CrossRefGoogle Scholar
  32. Hodkovicova N, Chmelova L, Sehonova P, Blahova J, Doubkova V, Plhalova L, Fiorino E, Vojtek L, Vicenova M, Siroka Z (2019) The effects of a therapeutic formalin bath on selected immunological and oxidative stress parameters in common carp (Cyprinus carpio). Sci Total Environ 653:1120–1127. CrossRefPubMedGoogle Scholar
  33. Hoseinifar S, Zoheiri F, Lazado C (2016) Dietary phytoimmunostimulant Persian hogweed (Heracleum persicum) has more remarkable impacts on skin mucus than on serum in common carp (Cyprinus carpio). Fish Shellfish Immunol 59:77–82. CrossRefPubMedGoogle Scholar
  34. Hoseinifar SH, Zou HK, Miandare HK, Van Doan H, Romano N, Dadar M (2017) Enrichment of common carp (Cyprinus carpio) diet with medlar (Mespilus germanica) leaf extract: effects on skin mucosal immunity and growth performance. Fish Shellfish Immunol 67:346–352. CrossRefPubMedGoogle Scholar
  35. Hoseinifar SH, Sohrabi A, Paknejad H, Jafari V, Paolucci M, Van Doan H (2019a) Enrichment of common carp (Cyprinus carpio) fingerlings diet with Psidium guajava: the effects on cutaneous mucosal and serum immune parameters and immune related genes expression. Fish shellfish immunol 86:688–694. CrossRefPubMedGoogle Scholar
  36. Hoseinifar SH, Zou HK, Paknejad H, Hajimoradloo A, Van Doan H (2019b) Effects of dietary white-button mushroom powder on mucosal immunity, antioxidant defence, and growth of common carp (Cyprinus carpio). Aquaculture 501:448–454. CrossRefGoogle Scholar
  37. Jia R, Liu B-L, Feng W-R, Han C, Huang B, Lei J-L (2016) Stress and immune responses in skin of turbot (Scophthalmus maximus) under different stocking densities. Fish shellfish Immunol 55:131–139. CrossRefPubMedGoogle Scholar
  38. Johnson C (2005) Clinical perspectives on the health effects of Moringa oleifera: a promising adjunct for balance nutrition and better health. KOS Health Publications, La CanadaGoogle Scholar
  39. Khalil F, Korni FM (2017) Evaluation of Moringa oleifera leaves and their aqueous extract in improving growth, immunity and mitigating effect of stress on common carp (Cyprinus carpio) fingerlings. Turk J fish Aquatic Sci 32:170–178CrossRefGoogle Scholar
  40. Kiemer MC, Black KD (1997) The effects of hydrogen peroxide on the gill tissues of Atlantic salmon, Salmo solar L. Aquaculture 153:181–189. CrossRefGoogle Scholar
  41. Krasnov A, Skugor S, Todorcevic M, Glover K, Nilsen F (2012) Gene expression in Atlantic salmon skin in response to infection with the parasitic copepod, Lepeophtheirus salmonis, cortisol implant, and their combination. BMC Genomics 13:130–145. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Livak K, Schmittgen T (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔ CT method. Methods 25:402–408. CrossRefGoogle Scholar
  43. Mansell B, Powell M, Ernst I, Nowak BF (2005) Effects of the gill monogenean Zeuxapta seriolae (Meserve, 1938) and treatment with hydrogen peroxide on pathophysiology of kingfish, Seriola lalandi Valenciennes, 1833. J Fish Dis 28:253–262. CrossRefPubMedGoogle Scholar
  44. Mansour AT, Miao L, Espinosa C, García-Beltrán JM, Francisco DC, Esteban MA (2017) The intestinal immune response (inflammation, tight junction protein and humoral immune genes expression) of gilthead seabream, Sparus aurata, fed moringa leaves supplemented diets. Paper presented at the Asian-Pacific Aquaculture 2017, July 24–27, Kuala LumpurGoogle Scholar
  45. Mansour A, Omar E, Srour T, Yousef M (2018a) Effect of three natural phytochemicals supplementation on growth performance, testosterone level and feed utilization of Nile tilapia (Oreochromis niloticus). Aquac Nutr 24:408–415. CrossRefGoogle Scholar
  46. Mansour AT, Miao L, Espinosa C, García-Beltrán JM, Francisco DCC, Esteban MÁ (2018b) Effects of dietary inclusion of Moringa oleifera leaves on growth and some systemic and mucosal immune parameters of seabream. Fish Physiol Biochem 44:1223–1240. CrossRefPubMedGoogle Scholar
  47. Mansouri TH, Hajimoradloo A, Hoseinifar S, Ahmadvand H (2017) Dietary Myrtle (Myrtus communis L.) improved non-specific immune parameters and bactericidal activity of skin mucus in rainbow trout (Oncorhynchus mykiss) fingerlings. Fish Shellfish Immunol 320–324. CrossRefGoogle Scholar
  48. Mohammed HH, Arias CR (2015) Potassium permanganate elicits a shift of the external fish microbiome and increases host susceptibility to columnaris disease. Vet Res 46:82. CrossRefPubMedPubMedCentralGoogle Scholar
  49. Muangnoi C, Chingsuwanrote P, Praengamthanachoti P, Svasti S, Tuntipopipat S (2012) Moringa oleifera pod inhibits inflammatory mediator production by lipopolysaccharide-stimulated RAW 264.7 murine macrophage cell lines. Inflammation 35:445–455. CrossRefPubMedGoogle Scholar
  50. Munne-Bosch S, Pinto-Marijuan M (2016) Free radicals, oxidative stress and antioxidants. Encycl Appl Plant Sci 2:16–19. CrossRefGoogle Scholar
  51. Murthy KS, Kiran B (2013) Review on usage of medicinal plants in fish diseases. Int J Pharm Bio Sci 4:975–986Google Scholar
  52. Ortuno J, Esteban M, Meseguer J (2000) Kinetics of hydrogen peroxide production during in vitro respiratory burst of seabream (Sparus aurata L.) head-kidney leucocytes, as measured by a flow cytometric method. Fish Shellfish Immunol 10:725–729. CrossRefPubMedGoogle Scholar
  53. Paliwal R, Sharma V, Pracheta SS (2011) Elucidation of free radical scavenging and antioxidant activity of aqueous and hydroethanolic extracts of Moringa oleifera pods. Asian J Biotechnol 4:566–571Google Scholar
  54. Quade MJ, Roth JA (1997) A rapid, direct assay to measure degranulation of bovine neutrophil primary granules. Vet Immunol Immunopathol 58:239–248. CrossRefPubMedGoogle Scholar
  55. Rach JJ, Gaikowski MP, Ramsay RT (2000) Efficacy of hydrogen peroxide to control parasitic infestations on hatchery-reared fish. J Aquat Anim Health 12:267–273.<0267:EOHPTC>2.0.CO;2 CrossRefGoogle Scholar
  56. Roque A, Yildiz HY, Carazo I, Duncan N (2010) Physiological stress responses of sea bass (Dicentrarchus labrax) to hydrogen peroxide (H2O2) exposure. Aquaculture 304:104–107. CrossRefGoogle Scholar
  57. Ross NW, Firth KJ, Wang A, Burka JF, Johnson SC (2000) Changes in hydrolytic enzyme activities of naive Atlantic salmon, Salmo salar, skin mucus due to infection with the salmon louse Lepeophtheirus salmonis and cortisol implantation. Dis Aquat Org 41:43–51CrossRefGoogle Scholar
  58. Sallam AE, Mansour AT, Srour TM, Goda AMA (2017) Effects of different carotenoid supplementation sources with or without sodium taurocholate on growth, feed utilization, carotenoid content and antioxidant status in fry of the European seabass, Dicentrarchus labrax. Aquac Res 48:3848–3858. CrossRefGoogle Scholar
  59. Sánchez NR, Ledin S, Ledin I (2006) Biomass production and chemical composition of Moringa oleifera under different management regimes in Nicaragua. Agrofor Syst 66:231–242CrossRefGoogle Scholar
  60. Sashidhara KV, Rosaiah JN, Tyagi E, Shukla R, Raghubir R, Rajendran SM (2009) Rare dipeptide and urea derivatives from roots of Moringa oleifera as potential anti-inflammatory and antinociceptive agents. Eur J Med Chem 44:432–436. CrossRefPubMedGoogle Scholar
  61. Shephard KL (1994) Functions for fish mucus. Rev Fish Biol Fisher 4:401–429. CrossRefGoogle Scholar
  62. Sies H (2017) Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress. Redox Biol 11:613–619. CrossRefPubMedPubMedCentralGoogle Scholar
  63. Sies H, Berndt C, Jones DP (2017) Oxidative stress. Annu Rev Biochem 86:715–748CrossRefGoogle Scholar
  64. Sreelatha S, Padma P (2010) Modulatory effects of Moringa oleifera extracts against hydrogen peroxide-induced cytotoxicity and oxidative damage. Hum Exp Toxicol 30:1359–1368. CrossRefPubMedGoogle Scholar
  65. Stohs SJ, Hartman MJ (2015) Review of the safety and efficacy of Moringa oleifera. Phytother Res 29:796–804. CrossRefPubMedPubMedCentralGoogle Scholar
  66. Subramanian S, MacKinnon S, Ross N (2007) A comparative study on innate immune parameters in the epidermal mucus of various fish species. Comp Biochem Physiol B Biochem Mol Biol 148:256–263. CrossRefPubMedGoogle Scholar
  67. Sundh HKB, Fridell F, Olsen RE, Ellis T, Taranger GL et al (2010) Intestinal barrier function of Atlantic salmon (Salmo salar L.) post smolts is reduced by common sea cage environments and suggested as a possible physiological welfare indicator. BMC Physiol 10:22. CrossRefPubMedPubMedCentralGoogle Scholar
  68. Swain P, Dash S, Sahoo P, Routray P, Sahoo S, Gupta S, Meher P, Sarangi N (2007) Non-specific immune parameters of brood Indian major carp Labeo rohita and their seasonal variations. Fish Shellfish Immunol 22:38–43. CrossRefPubMedGoogle Scholar
  69. Thibodeau P, Paquett B (1999) DNA damage induced by catechol estrogens in the presence of copper (II): generation of reactive oxygen species and enhancement by NADH. Free Radic Biol Med 27:1367–1369. CrossRefPubMedGoogle Scholar
  70. Van Doan H, Hoseinifar SH, Sringarm K, Jaturasitha S, Yuangsoi B, Dawood MA, Esteban MÁ, Ringø E, Faggio C (2019) Effects of Assam tea extract on growth, skin mucus, serum immunity and disease resistance of Nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae. Fish Shellfish Immunol 93:428–435. CrossRefPubMedGoogle Scholar
  71. Van Hai N (2015) The use of medicinal plants as immunostimulants in aquaculture: a review. Aquaculture 446:88–96. CrossRefGoogle Scholar
  72. Wang D, Li F, Chi Y, Xiang J (2012) Potential relationship among three antioxidant enzymes in eliminating hydrogen peroxide in penaeid shrimp. Cell Stress Chaperones 17:423–433. CrossRefPubMedGoogle Scholar
  73. Waterman C, Cheng DM, Rojas-Silva P, Poulev A, Dreifus J, Lila MA, Raskin I (2014) Stable, water extractable isothiocyanates from Moringa oleifera leaves attenuate inflammation in vitro. Phytochemistry 103:114–122. CrossRefPubMedPubMedCentralGoogle Scholar
  74. Whyte SK (2007) The innate immune response of finfish-a review of current knowledge. Fish Shellfish Immunol 23:1127–1151. CrossRefPubMedGoogle Scholar
  75. Zou J, Secombes CJ (2016) The function of fish cytokines. Biology 5:23–58. CrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Nature B.V. 2020

Authors and Affiliations

  • Abdallah Tageldein Mansour
    • 1
    • 2
    Email author
  • Cristóbal Espinosa
    • 3
  • Jose María García-Beltrán
    • 3
  • Liang Miao
    • 4
  • Diana C. Ceballos Francisco
    • 3
  • Ahmed Saud Alsaqufi
    • 2
  • M. Ángeles Esteban
    • 3
  1. 1.Fish and Animal Production Department, Faculty of Agriculture (Saba Basha)Alexandria UniversityAlexandriaEgypt
  2. 2.Department of Aquaculture and Animal Production, College of Agriculture and food SciencesKing Faisal UniversityAl HofufKingdom of Saudi Arabia
  3. 3.Fish Innate Immune System Group, Department of Cell Biology and Histology, Faculty of Biology, Regional Campus of International Excellence “Campus Mare Nostrum”University of MurciaMurciaSpain
  4. 4.Key Laboratory of Applied Marine Biotechnology, Ministry of EducationNingbo UniversityNingboChina

Personalised recommendations