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

1. 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
Md Mursalin Khan
Department of Biology
Indiana University-Purdue University Fort Wayne

2. OUTLINE
 Introduction
 Objectives
 In-vitro Study
 In-vivo Study
 Conclusion
 Impact of Research
 Future Research
 Acknowledgement

3. INTRODUCTION

4. BACKGROUND
 Animals along with human encounter various biological,
chemical, and physical stress from environment.
 Stress hampers animals’ homeostasis and causes the immune
response to decrease that prevents animals to fight against
the diseases.
 Diseased animals are treated with chemicals that are not good
for the animal health and for the environment.
 Researchers are currently studying nutraceuticals and natural
products to reduce stress and increase immune response in
animals including humans.

5. STRESS
 Stress
 Any stimulus that impairs the normal performance
 Chemical, physical, or biological
 Acute or chronic
 Levels of stress responses
 Primary: activation of neuroendocrine system and
production of stress hormones: cortisol (alarm)
 Secondary: effects of cortisol on physiological
systems (resistance)
 Tertiary: consequence of physiological changes
(exhaustion)
Ref: Mustafa, personal communication.

6. STRESS PHYSIOLOGY
Ref: Mustafa, Personal Communication. (Source: https://en.wikipedia.org/wiki/)

7. GENERAL ADAPTATION SYNDROME
(Source: keyword-suggestions.co(Source: Porchas et al. 2012)

8. NILE TILAPIA
 Fish has become a good model to study the stress because
the fish responses to stressors are higher than in the
terrestrial animals.
 Nile tilapia was used in the experiment as a member of the
hardy species which can handle most stress (such as low
oxygen, high ammonia), except for the crowding-related
stress.
(Source: www.eddmaps.o

9. ELDERBERRY(EB)
 Elderberry or Sambucus nigra has been used for centuries to
treat wounds and respiratory illnesses such as sinuses, cold,
and flu.
 Elderberry contains flavonoids, which have antioxidant
properties and may prevent damages to the cells.
 Elderberry have anti-inflammatory, antiviral, and anticancer
properties.
 Evidence suggests that chemicals in elderberry flower and
berries reduce inflammation in mucus membranes.
 However, very few studies have been done in humans, so
researchers don't know how effective elderberry can be.
(Sources: www.kansasforests.org, www.livestrong.com,

10. OBJECTIVES

11. RESEARCH OBJECTIVES
 The aim of our experiment was to determine the effect
of elderberry extract on the modulation of stress and
immune response in Nile tilapia in-vitro and in-vivo.

12. EXPERIMENTS IN-VITRO

13. In-vitro STUDY: OVERVIEW
 Mitogens were used as the stressor for the cells in the
culture media.
 In the thymidine incorporation assay, radioactive nucleoside,
3H-thymidine, was incorporated into new strands of
chromosomal DNA during mitotic (2n) cell division.
 A scintillation beta-counter was used to measure the
radioactivity in DNA recovered from the cells in order to
determine the extent of cell division.

14. Spleen or
Thymus
collection from
Nile tilapia
Single cell
suspension
preparation and
lymphocytes
count
1x106 cells,
RPMI 1640 with
10% FBS,
mitogens and EB
Incubate at 28
°C, 5% CO2 & 95%
humidity for
48hrs
Addition of
radioactive 3H-
thymidine
Incubate at 28
°C, 5% CO2 & 95%
humidity for
24hrs
Harvest cells and
transfer DNA in
the filter paper
Measurement of
radioactivity in
beta scintillation
counter
Comparison of
3H-thymidine
incorporation
PROLIFERATION ASSAY

15. T-CELL PROLIFERATION ASSAY
 T-cell Proliferation:
 Concanavalin A (Con A) is a lectin (carbohydrate-binding
protein) originally extracted from the jack-bean,
Canavalia ensiformis.
 Determination of effective concentration of mitogen
Concanavalin A (Con A) for spleen and thymus T-
lymphocytes proliferation.
 Quantification of T-lymphocytes proliferation in presence of
EB extract along with Con A [incorporating Radioactive 3H
Thymidine in the DNA of the proliferative T-lymphocytes].
www.ddw-online.com

16.  B-cells Proliferation:
 Lipopolysaccharides (LPS), also known as lipoglycans and
endotoxin, are large molecules consisting of a lipid and a
polysaccharide composed of O-antigen.
 Determination of effective concentration of mitogen
Lipopolysaccharides (LPS) for spleen B-lymphocytes
proliferation.
 Quantification of B-lymphocytes proliferation in presence of
EB extract along with Con A [incorporating Radioactive
Thymidine (3H) in the DNA of the proliferative T-
lymphocytes].
B-CELL PROLIFERATION ASSAY
www.directindustry.com

17.  Lymphokine Production:
 To measure the effect of EB extract on Interlukine-2
production from T-cells, in presence of mitogen Con A.
 Anti-IL-2 antibodies were allowed to bind with IL-2
released from spleen T-Helper cells and quantified using
Enzyme Linked Immune Sorbent Assay (ELISA).
ELISA FOR LYMPHOKINE PRODUCTION
www.bio-rad.com
phamnguyen.com.vn

18. EXPERIMENTAL DESIGN
 Proliferation of T-cells
 Control: T-cells (From Spleen or Thymus )
 Treatment 1: Spleen or Thymus T-cells + Con A
 Treatment 2: Spleen or Thymus T-cells + Con A +
Elderberry
 Proliferation of B-cells
 Control: B-cells (From Spleen)
 Treatment 1: Spleen B-cells + LPS
 Treatment 2: Spleen B-cells + LPS + Elderberry
 Production of Lymphokine (IL-2)
 Control: T-cells (From Spleen)
 Treatment 1: Spleen T-cells + Con A
 Treatment 2: Spleen T-cells + Con A + Elderberry

19. Spleen T cell proliferation result 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 than
control.
EB SHOWED HIGHER PROLIFERATION OF SPLEEN
T-CELLS
(Results are means ± SEM. Different than control: * = P<0.05, **= P<0.01, ***=
P<0.001)

20. Thymus T-cells need higher concentration (46.51 μg/mL and 88.88
μg/mL) to get the significant (P < 0.001) proliferation. 23.81 μg/mL
was unable to proliferate the thymus cells significantly like spleen T-
cells.
EB SHOWED HIGHER PROLIFERATION OF THYMUS
T-CELLS
(Results are means ± SEM. Different than control: * = P<0.05, **= P<0.01, ***=
P<0.001)

21. Spleen B-cell proliferation 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 to
the control
EB SHOWED HIGHER PROLIFERATION OF SPLEEN B-
CELLS
(Results are means ± SEM. Different than control: * = P<0.05, **= P<0.01, ***=
P<0.001)

22.  23.81 μg/mL elderberry
extract concentration
showed the highest
concentration rate
 46.51 μg/mL and 88.88
μg/mL showed less
proliferation than Con A
only
EB EXHIBITED HIGH IL-2
PRODUCTION
(Results are means ± sem. Different than control: * = P <0.05, **= P <0.01,
***= P <0.001) (Experimental replicates)

23.  Cell proliferation assay showed significant proliferation
of the spleen T and B-lymphocytes with the presence of
elderberry extracts.
 Cell proliferation assay for thymus T-lymphocytes also
provided the evidence of the T-cell proliferation by
elderberry extract.
 ELISA assay of lymphokine IL-2 production for the
spleen T-Helper cells provided necessary information
about the higher levels of IL-2 concentrations than
control.
SUMMARY (In-vitro Study)

24. EXPERIMENTS IN-VIVO

25. In-vivo: OVERVIEW
 Effects of elderberry supplementation (2% of the weight) in
feed for acute and chronic stress.
Physiological and immunological parameters are studied:
 Cortisol concentration using ELISA
 Blood glucose level
 Packed cell volume
 Total plasma protein
 Spleen somatic index
 Lysozyme activity against Micrococcus lysodeikticus
 Spleen lymphocytes count
 IL-2 production rate using ELISA.

26. In-vivo: OVERVIEW
 Nile tilapia fish were divided into CC = commercial feed,
control density (optimum); CEB = elderberry extract
supplemented commercial feed, control density (optimum);
StC= commercial feed, High density (stressed); StEB =
elderberry extract supplemented commercial feed, High
density (stressed) groups for “Acute (Day 1,3) and Chronic
(Day 7,14,21,28) Stress Study”.

27. PLASMA CORTISOL CONCENTRATIONS
 The Cortisol level was increased on Day 1 and Day 3 for the acute
stress.
 It remained similar on Day 7, but from Day 14 onwards, the cortisol
level started to decline for control density groups (CC and CEB).
 After Day 14, the data of Day 21 showed a marked decrease of
cortisol in the StEB when compared to StC. The trend was continued
to Day 28.
(Results are means ± SEM) (Experimental replicates)

28. BLOOD GLUCOSE MEASUREMENT: ACUTE
STUDY
 Stress groups has higher blood glucose level than control
groups.
 StEB group has lower glucose level than StC group which
indicate the stress reducing capability of elderberry extract.
Blood glucose level (means ± SEM), (P < 0.05) (e.g. a7 and b7 are significantly different on Day 7)

29. BLOOD GLUCOSE MEASUREMENT: CHRONIC STUDY
Blood glucose level (means ± SEM), (P < 0.05) (e.g. a7 and b7 are significantly different on Day 7)
 Day 7 data demonstrated significantly higher blood glucose
levels (P<0.05) in the stressed groups.
 The blood glucose levels of the elderberry supplemented
stressed group (StEB) were much lower on Day 14 (P<0.05)
compare to StC.

30. PACKED CELL VOLUME
Packed cell volume (means ± SEM), (P < 0.05)
 Due to the ambiguity of the PCV data among the groups it is
difficult to identify the role of elderberry extracts as a stress
reducing and immune stimulating factor.

31. TOTAL PLASMA PROTEIN
Total plasma protein concentrations (means ± SEM), (P < 0.05)
 From the total plasma protein data, the level of stress and
effect of the elderberry to reduce stress was not observed.

32. SPLEEN SOMATIC INDEX
Spleen somatic index (means ± SEM), (P < 0.05)
 Stress groups showed little higher index than CEB group.
But compared to the CC group, the relation was not
consistent.

33. SPLEEN LYMPHOCYTES COUNTS
 In the control density groups
(CC & CEB), spleen cell counts
were much higher than the
stressed groups (StC and StEB)
in acute stress condition
 CEB cell count was relatively
much higher compared to CC.
 On Day 7 cell counts went
down in the stresses groups
compared to CC and CEB.
 On Day 14, 21, and 28 the
spleen cell counts of the non-
supplemented stressed
groups (StC) remained lower
than other (CC, CEB, & StEB).
(Results are means ± SEM) (Experimental replicates)

34. LYSOZYME CONCENTRATIONS
Lysozyme concentrations (means ± SEM), (P < 0.05) (e.g. a7 and b7 are significantly different on Day 7)
 Higher levels of lysozyme concentrations were observed in the
acute stress and gradually decreased in the chronic stress for
stress groups (StC & StEB).
 On Day 28 data gave the evidence of the restoration the
equilibrium or homeostasis in the fish.

35. IL-2 CONCENTRATIONS
 Elderberry supplemented
group (StEB) are not capable
to produce higher level of
IL-2 compared to other
stressed group (StC).
 StC group showed high
concentration of the IL-2
than others throughout the
experimental Days (except
Day 1).
(Results are means ± SEM) (Experimental replicates)

36. CORRELATION: CORTISOL & GLUCOSE
 In the correlation study between cortisol and glucose we found
the trend of proportional increase.

37. CORRELATION: GLUCOSE & LYSOZYME
 The data analysis of the glucose and lysozyme revealed a
proportional increase of lysozyme concentration along with
elevated glucose level.

38. CORRELATION: CORTISOL & LYSOZYME
 The cortisol and lysozyme relationship showed relationship
between primary and tertiary in the stress physiology.

39.  Plasma cortisol levels increased during the acute stress phase
but gradually decreased during the chronic stress phase.
 Blood glucose levels were similar to plasma cortisol levels.
Cortisol and glucose levels were proportionally correlated.
 The spleen cell counts were lower in the stressed groups
compare to control groups. And much less in the non
supplemented stress group compare to elderberry
supplemented stress group.
SUMMARY (in-vivo)

40.  There were no significant variations among the groups for
lysozyme concentrations. The correlation between blood
glucose and lysozyme showed proportionate relation
between secondary and tertiary stress responses.
 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.
SUMMARY (in-vivo)

41. CONCLUSION

42.  Research showed the significant immune stimulation through
the T-cells and B-cells in the in-vitro conditions in the
presence of elderberry extract.
 The IL-2 production rate of the T-helper cells also showed the
potential of the elderberry extract as an immune stimulating
agent.
 Elderberry extract has the potential to increase the immune
response against mitogen Con A and LPS and high density
crowding stress.
 In the in-vivo systems, elderberry extract exhibited the stress
reducing capability.
 In-vitro and in-vivo study showed elderberry has the
medicinal properties to stimulate the immune response
against mitogens and high density crowding stress.
CONCLUSION

43. IMPACT OF RESEARCH
 Elderberry extract can be used as a natural medicinal
therapeutic to reduce stress and to stimulate immune
response.
 Elderberry will significantly reduce the use of chemical drugs
in aquaculture.
 It can also be used as a nutraceutical for reducing stress and
stimulating immune response in higher vertebrates including
humans in the future.

44. FUTURE RESEARCH
 Role of elderberry extract as a nutraceutical in other animal
models.
 Use of different stressors (such as low dissolved oxygen,
high or low temperature, high salinity, and handling) to
observe the effects of elderberry.
 Performe challenge studies to observed antiviral and
antibacterial activities of elderberry.

45. ACKNOWLEDGEMENT
 Major Supervisor: Dr. Ahmed Mustafa
 Co-supervisor: Dr. Elliott Blumenthal
 Committee Member: Dr. Jaiyanth Daniel
 Special Thanks: Dr. Robert Gillespie, Mrs. Karen McLellan, Mrs.
Marilyn Shannon, Dr. George Mourad, Dr. Mark Jordan & Dr.
Parveen Quarrar
 Graduate Students: Regina Shannon, Md Ibrahim, Tazin Fahmi &
Luna Wahab
 Undergraduate Students: Grayson Ostermeyer, Alycia Herndon,
Jessica Eash, Jesse Rinard & Destin Furnas
 Biology Department: Arlis Lamaster, Bruce Arnold, Carla Barrett,
Darlene Bender & Glenda Pray