Assessment of the role of elderberry extract in Nile tilapia, Oreochromis niloticus, as a stress reducing and immune stimulating nutraceutical in-vitro and in-vivo
Stress is a condition where the physiological demand exceeds the normal adjusting capacity of an organism and hampers the normal performance and immunity of the biological systems. There is no method or drug that can combat against stress with full success. Stress management is, therefore, one of the complex research sectors of the modern time. Use of nutraceuticals to reduce the stress and stimulate the immune system is a growing field of interest. Bioactive dark-pigmented elderberry extract has antioxidants, phenolic components, flavonoids, and anthocyanins which are capable of keeping the homeostasis of the cellular functions and has the potential to suppress tumor cell growth. The goal of our research was to examine the effect of elderberry extract on Nile Tilapia, on T and B cell response in vitro and then to modulate the physiological and immunological responses in-vivo. Four experimental groups of fish, Nile tilapia [two groups (Group I and II) were maintained at a low density (5 g/L) and two groups (Group III and IV) were at a high density (120 g/L) to induce a stress response. One each of the low and high density groups (Group II, CEB and Group IV, StEB) received elderberry supplemented commercial feed, and the other groups (Group I, CC and and Group III, StC) received the non-supplemented commercial feed) were used in-vivo for both acute and chronic stress.^ In the in-vitro study, spleen T-cell proliferation results showed significantly (P < 0.001) higher clonal proliferation rate in the presence of 23.81 μg/mL, 46.51 μg/mL, and 88.88 μg/mL elderberry extract concentrations than control in presence of Con A (2.38 μg/mL). Thymus T-cells proliferation result also showed significantly (P < 0.001) a higher clonal proliferation rate in the presence of 46.51 μg/mL, and 88.88 μg/mL elderberry extract concentrations only than control in presence of Con A (2.38 μg/mL). For thymus T-Cells, 23.81 μg/mL did not show any significant increase as previously shown in spleen T-cells. Spleen B-cell proliferation result supported the previously found results of the T-cells proliferation, mentioned above. It showed significantly (P < 0.001) higher proliferation rate in the presence of 23.81 μg/mL, 46.51 μg/mL, and 88.88 μg/mL elderberry extract concentrations when compared with the control in presence of LPS (2.38 μg/mL). Lymphokine IL-2 immunoassay showed significantly higher concentration of IL-2 synthesis when stimulated with 23.81 μg/mL, 46.51 μg/mL, and 88.88 μg/mL (P < 0.001) elderberry extract concentrations than control. But the production of the IL-2 did not exponentially increase. 23.81 μg/mL elderberry extract concentration showed the highest concentration rate, followed by 46.51 μg/mL and 88.88 μg/mL elderberry extract concentrations. ^ In the in-vivo study, plasma cortisol assay showed that the cortisol levels were increased for acute stress and gradually decreased in the chronic stress periods. The blood glucose results showed similar trend like plasma cortisol. In the correlation study between cortisol and glucose we found the trend of proportional increase. The spleen cell counts decline in the stressed groups. Lysozyme concentration results provided no significant variations among the groups but the correlation between lysozyme and blood glucose showed the proportional increase between secondary and tertiary stress responses. From the data of the IL-2 production assay, it is distinctly evident that elderberry supplemented groups were not capable to produce higher level of IL-2 compared to other stressed groups having no supplementation. But at the same time it is observed that stressed group having no supplementation (control) showed high concentration of the IL-2 than others throughout the experimental period.^ The significant clonal proliferation of the T-cells and B-cells in the in-vitro conditions and the higher rate of IL-2 production by the T-helper cells indicate the potential of the elderberry extracts as an immune stimulating factor. In the in-vivo conditions, elderberry extracts exhibited stress reducing capability during the acute stress but none during the chronic stress conditions, may be due to stress adaptability of Nile tilapia.^ With further research, elderberry extracts can be used as a supplementation in the feed in aquaculture for reduce stress and stimulate immune response in fish. It can also be used as a nutraceutical for reducing stress and stimulating immune response for higher vertebrates including humans in the future.^
Ahmed Mustafa, Purdue University.