EFFECT OF MEDICINAL PLANTS BHRINGARAJ (ECLIPTA ALBA) AND ALOE VERA (ALOE BARBADENSIS MILLER) METHANOL EXTRACT SUPPLEMENTED DIETS ON IMMUNITY AND DISEASE RESISTANCE AGAINST BACTERIUM AEROMONAS HYDROPHILA IN CIRRHINUS MRIGALA

1. FOURTH INTERNATIONAL CONFERENCE ON
AGRICULTURE, ANIMAL SCIENCE
AQUACULTURE & FISHERIES- 2016
Oral presentation
by
Mrs. P. Dhanalakshmi

2. EFFECT OF MEDICINAL PLANTS BHRINGARAJ (ECLIPTA ALBA) AND ALOE VERA (ALOE
BARBADENSIS MILLER) METHANOL EXTRACT SUPPLEMENTED DIETS ON IMMUNITY
AND DISEASE RESISTANCE AGAINST BACTERIUM AEROMONAS HYDROPHILA IN
CIRRHINUS MRIGALA
Under the supervision of
Dr. V. RAMASUBRAMANIAN., M.Sc., M. Phil., Ph.D.
Mrs. P. DHANALAKSHMI.
UNIT OF AQUATIC BIOTECHNOLOGY &
LIVE FEED CULTURE,
DEPARTMENT OF ZOOLOGY
BHARATHIAR UNIVERSITY

3. INTRODUCTION
1. World aquaculture has grown tremendously during the
past 20 years becoming an economically important field.
2. Aquaculture is one of the fastest growing food-
producing sector in the World. The increased intensification of
aquaculture has led to a increased number of disease outbreaks
with an high range of pathogens causing them (Kirubakaran et
al., 2010).

4. 3. Hence in recent years, increasing attention has been
given to the use of immunostimulants in the aquaculture
industry. Improvement of eco-friendly alternatives to
antibiotics that may keep fish healthy such as plant based
immunostimulants and probiotics has increased, indigenous
technological knowledge for controling diseases is enjoying
attention in disease control and fish health (Sahu et al., 2007).
4. The immunostimulation effects of plant medicines in
various fish species has been reported (Pugh et al., 2001).

5. OBJECTIVES
 The main objective of the study is to control the disease out
breaks in aquaculture farms using plant extracts.
 To determine the effect of plant extracts (E. alba and
A. vera) on the non-specific immune parameters of C. mrigala.

6.  To determine the efficacy of the plant extracts (E. alba and
A. vera) against the challenge pathogenic bacteria of
A. hydrophila -gram negative bacteria.
 To evaluate the effect of plant extracts on the immune response
and survival of C. mrigala fingerlings after the challenge of
A. hydrophila- gram negative bacteria.

7. MATERIALS AND METHODS
In the present study, fresh water fish C. mrigala was used as an
experimental organism to study the effect of plant extracts against
A. hydrophila.
FIGURE-1. CIRRHINUS MRIGALA (MRIGAL CARP)

8. CLASSIFICATION
Classification of Mrigal carp
• Kingdom - Animalia
• Phylum - Chordata
• Class - Actinopterigii
• Order - Cypriniformes
• Family - Cyprinidae
• Genus - Cirrhinus
• Species - mrigala
• Binomial name - Cirrhinus mrigala
• Bloch(1795)
• Comman name – mrigal carp
A) Eclipta alba (Bhringaraj)
• Kingdom - Plantae
• Division - Magnoliophyta
• Class - Magnoliopsida
• Order - Asterales
• Family - Asteraceae
• Genus - Eclipta L.
• Species – alba(L)
• Botanical name - Eclipta alba
• Popular name - Karisha-langanni,
Karisirang-kanni, Babri, and
Kesuri.

9. B) Aloe vera
• Kingdom - Plantae
• Clade-Angiosperms
• Clade-Monocots
• Order - Asparagalessz
• Family - Xanthorrhoeaceae
• Sub family - Asphodeloidae
• Genus - Aloe
• Species - vera
• Binomial name - Aloe vera(L)
(Burn. F.)
B) Aeromonas hydrophila
Domain - Bacteria
Kingdom – Proteobacteria
Phylum - Gamma proteobacteria
Class - Aeromonadales
Genus – Aeromonas
Species – hydrophila
Binomial name – Aeromonas
hydrophila
Chester (1901)
Stanier (1943)

10. PREPARATION OF PLANT EXTRACTS
Plant extract A: (E. alba)
The powdered aerial parts (15g) of E. alba were extracted
separately to exhaustion in a soxhlet apparatus using 99%
methanol (150 ml).
The extract were filtered through a cotton plug followed
by what man filter paper and then concentrated by using a rotary
evaporator at low temperature (40o-50oC).
The extract were preserved in airtight containers and kept
at 4o c until further use.

11. FIGURE-2. A VIEW OF ECLIPTA ALBA
FIGURE- 3. CLEAN ECLIPTAALBA PLANTS & LEAVES

12. FIGURE- 4. AERIAL PARTS OF ECLIPTA FIGURE-5. ECLIPTAALBA POWDER
ALBA IN DRIED FORM
FIGURE- 6. SOXHLET APPARATUS
WITH EXTRACT
FIGURE- 7. ECLIPTA ALBA PLANT
EXTRACT MIXED FEED

13. Plant extract B: (A. vera)
The A. vera leaves were sterilized properly. Fresh A. vera
leaves with gel was dried in the oven at 40oc for 48 hours and then powdered.
In the process of maceration, 15g of crushed plant part was dissolved in 150ml
of organic solvent i.e. methanol. The conical flask was covered by cotton plugs
to avoid solvent evaporation.
The extract was placed in shaking incubator at 250 rpm for 48 hours.
After shaking, it was filtered with muslin cloth. The filtered extract was
centrifuged at 8000 rpm for 20 minutes. The supernatant was collected in
sterile flask. Then, it was stored at 40c.

14. FIGURE- 8. A VIEW OF ALOE VERA PLANT
FIGURE- 9. A CLEAN ALOE VERA & ALOE VERA PIECES

15. FIGURE-10. ALOE VERA PIECES IN DRIED FORM
FIGURE-11. ALOE VERA POWDER

16. FIGURE-12. ALOE VERA PLANT EXTRACT FIGURE-13. ALOE VERA PLANT EXTRACT MIXED FEED

17. EXPERIMENTAL FOOD
PREPARATIONFor group 1
Normal balanced feed composed of 42% fish meal, 20% groundnut oil cake
powder, 15% tapioca powder, 15% wheat flour, 5% egg white and 3% mineral-
Vitamin mixture was used as control diet (protein: 39%, carbohydrate: 24% lipid:
11% and ash: 9%).
Group 2
The diet used in the experiment group 2, were prepared by mixing commercial
carp food with the methanol extract of E. alba in ratio 5g of E. alba per kilogram of
food. (i.e, 0.5 % E. alba).
Group 3
The diet used in the experiment group 3 were prepared by mixing commercial
carp food with the methanol extract of A. vera in ratio 5g of A. vera per kilogram of
food. i.e, 0.5% A. vera.

18. EXPERIMENTAL SETUP
Group 1: fishes were fed with
200 mg of control feed.
Group 2: fishes were fed with 200
mg of E. alba mixed feed
Group 3: fishes were fed with 200 mg of
A. vera mixed feed

19. SAMPLING
SCHEDULE
0’day - 1st blood
sample collection.
15thday - 1st
infectious
challenge with
A. hydrophila.
30thday - 2nd blood
sample collection.
35th day - 2nd
infectious challenge
with A. hydrophila.
45thday - 3rd blood
sample collection.

20. LYSOZYME ACTIVITY
Serum lysozyme activity was measured by adapting the method described
by (Parry et al., 1965).
SERUM BACTERICIDALACTIVITY
The method used for serum bactericidal activity was followed a modified
version of that adopted by (Kajita et al., 1990).
TOTAL SERUM PROTEIN
Samples were analyzed for total protein using the method outlined by
(Lowry et al. 1951).
SERUM GLOBULIN
Serum albumin content was measured using a standard albumin estimation
kit and the globulin content was estimated by subtracting albumin from total
protein.

21. HEMATOLOGY
LEUKOCYTE COUNT
Leukocytes (WBC) were counted by the method of (Rusia and Sood 1992) using
haemocytometer.
RED BLOOD CELL COUNT
Red blood cell (RBC) were determined as described by (Schaperclaus et al., 1991).
PACKED CORPUSCULAR VOLUME (PCV):
The packed cell volume (PCV) was determined by centrifugation at 2000 rpm for 20 min.
STATISTICAL ANALYSIS
The data’s were statistically evaluated by One-Way analysis of variance (ANOVA)
followed by Post Hoc multiple comparison test using SPSS software. The levels of
significance P < 0.05 was considered as statistically significant and P < 0.01 as highly
significant.

22. RESULTS
Fig: 14. Serum lysozyme (U/ml-1) count for C. mrigala fed same
dosages (200mg) of E. alba and A. vera at different days. Data
are represented as mean ± SD, n=4
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
Day-0 Day-15 Day-30 Day-45
control
Eclipta alba
Aloe vera

23. Fig.15. Serum bactericidal activity for C. mrigala fed same
dosages (200mg) of E. alba, A. vera at different days. Data are
represented as mean ± SD, n=4.
0
20
40
60
80
100
120
140
160
180
200
Day-0 Day-15 Day-30 Day-45
control
Eclipta alba
Aloe vera

24. Fig.16. Serum protein (g/dl) level for C. mrigala fed same
dosages (200mg) of E. alba, A. vera at different days. Data are
represented as mean ± SD, n = 4.
0
0.5
1
1.5
2
2.5
Day-0 Day-15 Day-30 Day-45
control
Eclipta alba
Aloe vera

25. Fig.17. Serum globulin (g/dl) level for C. mrigala fed same
dosages (200mg) of E. alba, A. vera at different days. Data are
represented as mean ± SD, n = 4.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Day-0 Day-15 Day-30 Day-45
control
Eclipta alba
Aloe vera

26. Fig.18. Leukocyte count (/mm3) for C. mrigala fed same dosages
(200mg) of E. alba, A. vera at different days. Data are
represented as mean ± SD, n = 4.
0
5
10
15
20
25
30
35
40
45
Day-0 Day-15 Day-30 Day-45
control
Eclipta alba
Aloe vera

27. Fig.19. Red blood cell count (×106 cells /mm3) for C. mrigala fed
same dosages (200mg) of E. alba, A. vera at different days.
Data are represented as mean ± SD, n = 4.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Day-0 Day-15 Day-30 Day-45
control
Eclipta alba
Aloe vera

28. Fig.20. The packed cell volume (PCV - %) for C. mrigala fed
same dosages (200mg) of E. alba, A. vera at different days.
Data are represented as mean ± SD, n = 4
23
23.5
24
24.5
25
25.5
26
26.5
27
27.5
28
28.5
Day-0 Day-15 Day-30 Day-45
control
Eclipta alba
Aloe vera

29. Fig.21 . Rate of mortality (%)
0%
5%
10%
15%
20%
25%
30%
Day-4 Day-8 Day-12 Day-14
percentageofmortality
Mortality rate
control
Eclipta alba
Aloe vera

30. FIGURE-22. NUCLEATED FISH RBC

31. FIGURE-23. A) CULTURE OF THE PATHOGENIC BACTERIA AEROMONAS HYDROPHILA
B) MICROSCOPIC VIEW OF AEROMONAS HYDROPHILA

32. DISCUSSION
 Application of immunostimulators, particularly plant immunostimulants
in the fish culture industry, can be considered a remarkable advantage
because of their safety and the fact that they are considered
environmentally friendly.
 In the present study, oral administration of A. vera and E. alba increased
serum lysozyme activity compared with control groups .
 In this study, the serum bactericidal activity increased significantly
(p < 0.05) in A. vera and E. alba treated groups when compared with
control group However, the presence of anti A. hydrophila antibody in
the treated fish could be the cause for the increased bactericidal activity.

33. In this study in C. mrigala, serum protein and globulin content were
significantly (P< 0.05) increased after oral administration of E. alba
and A. vera, when compared with control group.
The present study indicates that there was in increase in TLC in the
E. alba and A. vera supplemented groups while control group
showed depressed level of leukocyte count.
After the fingerlings were intra peritoneally challenged with
A. hydrophila. This is mainly due to the immune response of the
fish immune system against the bacterial invasion. The gradual
reversion of the leukocyte count back to the normal may be
indicative of recovery from systemic damage.

34. In this study, administration of A. vera and E. alba in
treated groups enhanced the survival rate after a challenge
with live A. hydrophila.
The highest survival rate in A. vera (80%), and E. alba
(70%) were observed respectively.
In this study, significant (P <0.05) differences were
observed in RBC count and PCV-values of the E. alba and
A. vera treated groups compared with control group after
challenge with A. hydrophila in mrigal carp.

35. CONCLUSION
The present results have demonstrated that the oral
administration of E. alba and A. vera can enhance the
specific and non- specific immune responses. The results
reported that a 0.5% E. alba and A. vera supplementation
can increase the resistance against A. hydrophila in
C. mrigala.

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39. Acknowledgements
I am thankful to my respected guide Dr. V.
Ramasubramanian M.Sc., M. Phil., Ph.D. Associate professor,
Department of Zoology, Bharathiar University for his valuable
guidance, continuous encouragement and constant support in my
research work.
I express my sincere thanks to Department of Microbiology,
PSG-IMSR, Coimbatore, Tamil Nadu, India, for providing the
microorganisms.