ON CLARIAS BATRACHUS FISH INFECTED WITH AEROMONAS ... · The Total erythrocyte count and total...
Transcript of ON CLARIAS BATRACHUS FISH INFECTED WITH AEROMONAS ... · The Total erythrocyte count and total...
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EFFECT OF AQUEOUS LEAF EXTRACT OF ANNONA SQUAMOSA
ON CLARIAS BATRACHUS FISH INFECTED WITH AEROMONAS
HYDROPHILA WITH REFERENCE TO HAEMATOLOGICAL
PARAMETERS.
Runa Paul*1, Asha Khanna
2, Daya Shankar Gautam
1 Rita Bhandari
3, Divya Patel
1
Pranjul Nigam1
1Department of Zoology,
St. Aloysius (Autonomous) College, Jabalpur. (M.P).
2Department of Zoology & Biotechnology, Govt. P.G Science College, Jabalpur. (M.P).
3Department of Zoology, Govt. OFK Degree College Khamaria, Jabalpur. (M.P).
ABSTRACT
Fish culture plays a vital role in fulfilling demand of human. In India
fresh water fishes are prone to bacterial infection. To increase the
productivity of healthy fishes the bacterial infected fishes are treated
with an antibiotic which cure the disease but have an adverse effect
also. The plant parts are usually considered as harmless and can be
used as a good replacement for antibiotics. Annona squamosa leaf is
well known for its antibacterial property. The current study was
conducted to investigate the effect of aqueous extract of leaf of Annona
squamosa in fresh water fish Clarias batrachus challenged with Aeromonas hydrophila.
Fishes (weight 110-160g) were divided into four groups (n=21). The fishes of control and
experimental groups I, II III & IV were bathed in various concentration of (5ml) 25%, (10ml)
50% and (15ml) 100% of 15ml of leaf extract in 15litre of aquarium water for 30 days and
then challenged with A. hydrophila. After 30 days of exposure of fishes in control and
experimental groups, haematological parameters were evaluated. They were post-challenged
with Aeromonas hydrophila and again haematological parameters was analysed after 15 days.
It was observed that the fishes bathed in various concentrations and fed with supplemented
diet showed decline in the total erythrocyte counts and haemoglobin content after infection
irrespective of the administration of bath dose. However, experimental fishes exposed in
100% concentration of aqueous leaf extract of Annona squamosa showed increase in
haemoglobin. Further it was observed that fishes exposed in 25%, 50% and 100%
World Journal of Pharmaceutical Research SJIF Impact Factor 7.523
Volume 7, Issue 1, 992-1005. Research Article ISSN 2277– 7105
Article Received on
14 Nov. 2017,
Revised on 04 Dec. 2017,
Accepted on 24 Dec. 2017
DOI: 10.20959/wjpr20181-10535
*Corresponding Author
Runa Paul
St. Aloysius (Autonomous)
College, Jabalpur. (M.P).
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Runa et al. World Journal of Pharmaceutical Research
concentration of aqueous leaf extract of Annona squamosa demonstrated increased in total
leucocyte counts (TLC) and Differential leucocyte counts in infected fishes which can be
regarded as a sign of improvement in immune response. Thus it was observed that after the
application of various concentration 25%, 50% and 100% of aqueous leaf extract of Annona
squamosa in experimental fishes can act as an immunostimulator.
KEYWORDS: Aeromonas hydrophila, Immunostimulant, Annona squamosa, Clarias
batrachus, Haematology.
INTRODUCTION
Fish culture is an old practice in India, which is the second largest culture fish producer in the
world.[1]
Fishes not only play an important role in the demand of food for humans but they
are widely used for various biological experiments.[2]
Bacterial infections are the major
reasons for fish mortality in aquaculture industry.[3]
Aeromonas hydrophila is a gram negative
opportunist bacterium associated with aquatic animal disease.[4]
A.hydrophila causes mass
mortalities in several species including Carps, Snake heads, Gouramies and Cat fishes and are
considered as an etiological agent of several diseases such as emaciation, haemorrhagic,
septicaemia, asymptomatic septicaemia, ulcerative infection, tail rot and fin rot.[5]
However,
the use of antibiotics in aquaculture poses threats such as development of bacterial strains that
are resistant to antibiotic treatment or the occurrence of antibiotic residues in fish farmed for
human consumers.[6]
In India no commercial vaccine or recommended immunotherapy is
currently available for catfish culture.[7]
The plant products are generally regarded as
harmless and can be used as novel methods of minimizing disease risk and as a good
substitution for antibiotics in aquaculture.[8]
Annona squamosa, sugar apple is native to the
tropic America and is widely grown throughout the tropics in India.[9]
Phytochemical studies
on Annona squamosa showed many active compounds which are having many
pharmacological activities such as anti-inflammation, anti-tumour activities.[10]
Studies have
shown that the leaves of Annona species are used as a vermicide, for treating cancerous
tumors and are applied to abscesses, insect bites and other skin complaints. Scrapings of root-
bark are used for toothache. The leaves and bark of custard apple contains alkaloids and the
fruit contains iron, calcium, fiber, amino acids, vitamins, carotene, thiamine, riboflavin,
niacin and ascorbic acid.[11]
It was observed that the different solvents extract of Annona
squamosa leaf confirmed antibacterial activity and presence of sterols, flavonoids, tannins in
various extracts was also observed.[12]
Clarias batrachus is the Asian catfish, locally known
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as ‘magur’ is a favourite edible fish in India and other Asian countries including Bangladesh,
Thailand, Vietnam, Malaysia and Indonesia.[13]
Intensive culture of Clarias batrachus is done
in many states, since it requires no special treatment and growth factors for culture, as such
work on immunostimulatory aspects in this fish will serve to increase the fish production and
help in solving the national food problem to some extent.[14]
Hence, the aim of the study was to determine the efficacy of aqueous leaf extract of Annona
squamosa, used as bath treatment to observe the effect on haematological parameters of
freshwater fish Clarias batrachus after pre and post challenged with bacterial pathogen
A.hydrophila.
MATERIALS AND METHODS
Collection acclimatization and maintenance
Clarias batrachus fishes of mixed sexes weighing about 110gm-160gm was used for the
study which was purchased from the local fisherman of Jabalpur, Madhya Pradesh, Indiaand
was identified with the help of the plate XI fig.3(fig.140) pp. 305 of the book cat fishes of
India by K.C Jayaram (2006). Fishes were disinfected with potassium per magnet. Fishes
were kept in aquarium capacity of 100l and allowed to acclimatize to laboratory conditions
for 15 days with continuous aeration. During the experiment the water was maintained at a
temperature of 27oC - 28
oC, pH at 7.5, D.O level of (6.8–7.2 mg/L), Alkalinity mg/L(85.4 -
97.6), Hardness mg/L (44-56). Ammonia and nitrite levels in the water were 0.00 mg/L.
Collection of Plant Material and Authentication of the plant
The leaves of the Annona species was collected from St. Aloysius College (Autonomous),
Jabalpur garden then Annona species was identified and authenticated by a Professor of
Botany Dr. Shailendra Tiwari, Senior Scientist State Forest Research Institute (SFRI),
Jabalpur. The leaf was washed thoroughly first in tap water and then rinsed with distilled
water and dried completely in shade at room temperature for 30 days. The plant material was
crushed and blended to fine powder in an electronic grinder (mixer) and stored in air tight
plastic container till further use.
Preparation, screening and antibacterial activities of the plant extracts
For aqueous extraction, 5g of the leaf powder were taken in 500ml distilled water and boiled
in water bath at 50-60°C for two hours. It was then filtered through Whatman filter paper no.
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1. The crude extracts were then stored in screw capped bottles in refrigerator at 4°C for
further use.
The qualitative assays of prepared plant extracts were used to analysed for the presence of
alkaloids, tannins, saponins, flavonoids, glycosides, phenolic compounds, terpenoids and
steroids.[15]
Experimental Design
Fishes of mixed sexes were divided into four groups (n = 21) and kept in glass aquarium
containing 15 litre of water. The control and experimental groups I, II & III were exposed to
5,10 and 15ml of dose in 15Literof different concentration of (25%, 50%,100%) aqueous leaf
extract of Annona squamosa in it every alternate day till 30 days. The control group and
experimental fishes were fed with pelleted commercial food and worms at 2% of body weight
and water was changed daily after 24 hours of feeding. Growth rate and haematological
parameters were determined after 15 and 30 days. After 30 days of immersionin different
concentration of aqueous extract of leaf, all experimental fishes were taken out and were kept
in water devoid of leaf extract and then all experimental groups were given only control feed
mentioned above at the rate of 2% of body weight. At the 30th
day of immunomodulation, the
fishes of experimental groups were challenged with Aeromonas hydrophila intramuscularly
in the caudal region at a dose of 0.5ml of 1.5×107cells/ml. After 15 days of infection, studies
were carried to monitor the growth rate and haematological parameters.
Determination of haematological parameters
Blood sample was collected from caudal vein before feeding in morning hours from
randomly picked fishes from control and experimental groups and then was transferred to vial
coated with anticoagulant EDTA to avoid coagulation and following hematological
parameters were investigated, hemoglobin estimation was done by Cyanmethemoglobin
method[16]
and the value is expressed in g%. The Total erythrocyte count and total leucocyte
count were determined by using a Neubauer's haemocytometer[17]
with slight modification
and differential leucocyte counts were counted by staining blood smears by May
Grunewald’s and Giemsa stain.
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RESULTS
Phytochemicals screening study
Table-1: Phytochemicals screening of Annona squamosal.
Phytochemicals Results
Alkaloids +
steroids +
phenol +
carbohydrate +
flavonoids +
saponin +
Reducing sugar +
Tannins +
Glycosides +
Our investigation showed that aqueous leaf extracts of the Annona squamosa Linn. Plants
contain considerable amounts of most of the secondary metabolites like alkaloids, steroids,
tannins, phenols, reducing sugar, saponin and flavonoids.
Effect of aqueous leaf extract of Annona squamosa on haemoglobin level of Clarias
batrahus.
It was observed that the haemoglobin content was found higher in non-injected treated fishes
(Gr.II, III & IV) in various concentration of aqueous extract of Annona squamosa as
compared to control Gr.I fishes.7.2±0.2, 7.83±0.15, 8.17±0.06 and 8.37±0.06(at 0 days),
7.5±0.1, 8.1±0.1,8.3±0.06 and 8.8±0.2 (at 15 days),7.6±0.2, 8.46± 0.12, 8.53 ± 0.12 and 9.5 ±
0.2 (at 30days) shown in Table 3. The haemoglobin content of injected fish with 0.5ml of
1.5×107cells/mL (Gr. II, III & IV) were towards higher side as compared to control Gr. I but
showed minimal decrease as compared to non-injected fishes 6.4±0.37,6.76±0.20, 6.9±0.1
and 7.1±0.1may be due to stress shown in Table 3 & 4.
Table-3: Shows the effect of Annona squamosa leaf extract on haemoglobin level in
Clarias batrachus fish after 0, 15 and 30 days.
Days
Treatment/Bath Doses
0days
g/dl
15 days
g/dl
30days
g/dl
ControlGroup I 7.2±0.2 7.5±0.1 7.6±0.2
Group II (25%) 7.83±0.15 8.1±0.1 8.46±0.12
Group III(50%) 8.17±0.06 8.3±0.06 8.53±0.12
Group IV(100%) 8.37±0.06 8.8±0.2 9.5±0.2
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Table-4 Show the effect of Annona squamosa leaf extract on haemoglobin level of
Clarias batrachus fish challenged with A.hydrophila after 15 days.
Days
Treatment/
Doses
Treatment 15days
Control Group I Post challenged (I/M)
with (A.hydrophila)
at a dose of 0.5ml
6.4±0.37
Group II (25%) 6.76 ±0.20
Group III(50%) 6.9±0.1
Group IV (100%) 7.1±0.1
Effect of aqueous leaf extract of Annona squamosa on TEC & TLC counts of Clarias
batrahus.
Total Erythrocyte Count (TEC) was found to be high in Gr. II, III & IV as compared to
control Gr. I, the mean values are 2.519±0.28, 2.530±0.25, 2.556±0.25 and 2.490±0.26,
respectively (at 15days). 2.529±0.26, 2.557±0.26, 2.587±0.26 and 2.524±0.25, respectively
(after 30days). The result shows thatthe TEC countwas increased in uninfected fish at higher
concentration of leaf extract. The total erythrocyte count was on lower side in experimental
fishes after 15 days challenged with A. hydrophila. The total erythrocyte count in Control
Gr.I, Gr II, Gr.III and Gr.IV was found to be 2.503±0.35, 2.517±0.31, 2.547±0.24 and
2.564±0.24, respectively. The TEC count values showed minimal decreased in post-
challenged group as compared to pre- challenged group after 15 days could be due to stress
shown in Table 5&6.
TLCs value has been showed an increasing trend in all experimental groups exposed to
various concentration of aqueous extract of Annona squamosa when compared to control
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group. The highest mean value of WBC (4318±92) was observed in Gr.IV and the lowest
value in control Gr.I.
After the 15th
day of infection, the highest value of TLC count (4325±115) was seen in Gr.IV
exposed in 100% aqueous leaf extract of Annona squamosa and the TLC count was lowest
(3890±101) in the control fish shown in Table 5&6.
Table – 5: Effect of aqueous leaf extract ofAnnona squamosa in TEC and TLC of fishes
after 15 and 30 days and post challenged with Aeromonas hydrophila after 15 days.
Table – 6: Effect of aqueous leaf extract ofAnnona squamosa in TEC and TLC of fishes
post challenged with Aeromonas hydrophila after 15 days.
TEC and TLC 15 days 30 days
Doses of leaf
extract %
Group I
(Control) 0
Group II
25
Group III
50
Group IV
100
Group I
(Control) 0
Group II
25
Group III
50
Group IV
100
TEC (million/cu.
mm) 2.490±0.26 2.519±0.28 2.530±0.25 2.556±0.25 2.524±0.25 2.529±0.26 2.557±0.26 2.587±0.26
TLC (cells/cu.
mm of blood) 3760±66 3981±202 4057.33±68 4181±57 3798.33±99 4035.20±268 4136±135 4318±92
TEC and TLC After 15 days post challenged with Aeromonas hydrophila
Doses of leaf extract % Group I
(Control) 0
Group II
25
Group III
50
Group IV
100
Doses of bacteria ml 0.5 0.5 0.5 0.5
TEC (million/cu.mm) 2.503±0.35 2.517±0.31 2.547±0.24 2.564±0.24
TLC (cells/cu.mm of blood) 3890±101 4163.33±194 4210±105 4325±115
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Effect of aqueous leaf extract of Annona squamosa on Differential Leucocyte Count of
Clarias batrahus
A differential leucocyte count was found to be abundant in all the experimental groups. The
total count of lymphocytes of control fish had a mean value of 45.6±2.1% whereas group I, II
& III had 49.33±5.1%, 52.33±10% & 54.33±13% at 15 days and 48.66±4.04%, 53±11%,
57±18% & 60.33±24% at 30days. Whereas the granulocytes values were 11±112±1, 3.33±0.6
& 13.67±1.2% at 15 days and 12.66±0.6%, 13.66±2.1%, 14±2.6% & 14.33±2.3% at 30 days.
In monocytes the mean values were as follows 6.33±1.5%, 7±2.6%, 7.66±3.7% & 8±4.3% at
15 days and 7±2.6%, 8.33±4.04%, 9.33±5.8% & 9.66±6.4% at 30days. The highest count of
lymphocytes followed by granulocytes and monocyte was seen in fish exposed in 100%
aqueous leaf extract concentration as compared to control group. After the 15th
day of
infection, the highest count of lymphocytes of 63.66±36%, granulocytes 15.66±3.5% and
monocytes 10.66±4.04% was obtained from the fish exposed in 100% aqueous leaf extract of
Annona squamosa and the lowest count of lymphocytes of 49.33±12%, granulocytes
13.33±2.5% and monocytes 7.66±0.57% was obtained in the control fish on the same day
shown in Table 7& 8.
Table- 7: Effect of aqueous leaf extract of Annona squamosa on lymphocytes, monocytes
and granulocytes of Clarias batrachus after 15 and 30 days.
Group/ Doses of
extract %
15 days 30days
Lymphocytes
% Monocytes%
Granulocytes
%
Lymphocytes
%
Monocytes
%
Granulocyte
s%
Control Group I 45.6±2.1 6.33±1.5 11±1 48.66±4.04 7±2.6 12.66±0.6
Group II (25%) 49.33±5.1 7±2.6 12±1 53±11 8.33±4.04 13.66±2.1
Group III(50%) 52.33±10 7.66±3.7 13.33±0.6 57±18 9.33±5.8 14±2.6
Group IV (100%) 54.33±13 8±4.3 13.67±1.2 60.33±24 9.66±6.4 14.33±2.3
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Table-8: Effect of Aqueous leaf extract of Annona squamosa on lymphocytes, monocytes
and granulocytes of Clarias batrachus after 15 days challenged with Aeromonas
hydrophila.
Group/ Doses of
extract %
15 days
Lymphocytes % Monocytes% Granulocytes%
Control Group I 49.33±12 7.66±0.57 13.33±2.5
Group II (25%) 54.66±21 8.66±2.1 14±2
Group III (50%) 58.66±28 9.66±3.7 14.66±2.3
Group IV (100%) 63.66±36 10.66±4.04 15.66±3.5
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DISCUSSION
Natural immnuostimulants are biocompatible, biodegradable and safe for the environment
and human health.[18]
These products can be used as novel methods of minimizing disease
risk and as a good substitution for antibiotics in aquaculture.[8]
Our results depicted that the
fish bathed in 100% leaf extract of Annona squamosa showed increase in the TEC count,
haemoglobin level, TLC count and Differential Leucocyte count in pre-challenged and post
challenged groups, but it was observed that the TEC count and haemoglobin level showed
minimal decrease in post challenged group as compared to pre challenged group after 15 days
could be due to stress. It was also found that aqueous leaf extracts of the Annona squamosa
Linn. plants contain considerable amounts of most of the secondary metabolites like
alkaloids, steroids, tannins, phenols, reducing sugar, saponin and flavonoids.
Neethu Simon K et al., (2016)[19]
also found that the leaf extracts of Annona squamosa
showed high antibacterial activity against negative bacterial strains than anti-fungal activity
and are also rich source of valuable primary and secondary metabolites. Samidha M.
Pawaskar et al., (2017)[20]
reported that the aqueous extract of leaf contains alkaloids, tannins,
saponins, flavonoids, glycosides and phenolic compounds and they also concluded that the
leaf extracts of Annona squamosa Linn. in all the different solvents used for extraction,
possesses potential antimicrobial activity against the pathogens for both gram positive &
gram negative bacterial strains.
Phytochemicals and antibacterial activity screening of the leaf and seed in water, methanol,
chloroform, petroleum ether and hexane was done by Gowdhami et al. (2014).[21]
The results
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indicated that the methanol and water extracts of seed and leaf had more positive results for
alkaloids, oils, tannins, phenols and flavonoids and also showed good inhibition of bacterial
strains (Gram negative origin) as compared to the standard antibiotics.
The current study agrees with the results of A. Sivagurunathan et al.,(2014)[22]
reported that
cinnamon diet fed fishes exhibited challenged with Pseudomonas aeruginosa showed decline
in the total erythrocyte counts and haemoglobin concentration after infection as compared to
uninfected group, increase in total leucocyte counts (TLC) and lymphocyte counts in infected
fishes. Sivagurunathan et al., (2011)[23]
similar results were observed in Cirrhinus mrigala
infected with Pseudomonas aeruginosa i.e., decrease in TEC & Hb.
After 50 days’ of administration of leaf extract of Euphorbia hirta in Cyprinus carpio fish by
Vijayakumari Pratheepa et al.,(2014)[24]
showed that haemoglobin content and TEC count
were decreased in challenged fish and on the 15th day onwards the haemoglobin level and
TEC count were increased in all the fish.
Mohsen Abdel-Tawwab et al., (2010).[25]
The obtained results have the same view in Nile
Tilapia fed with green tea infected with Aeromonas hydrophila showed an increase in WBC
and Lymphocyte counts. The same result was seen in T. Annalakshmi et al.,(2013).[26]
Labeo
rohita fed with feed supplemented with Phyllanthus aramus and infected with A.hydrophila
shown an increase in WBC, neutrophil, lymphocyte and monocyte.
Similar result was obtained by Pratheepa V et al.,(2010)[27]
in the efficacy of different dietary
doses of Aegle marmelos leaf extract in fish, Cyprinus carpio infected by Aeromonas
hydrophila after 50 days of feeding trial and the disease resistance of Cyprinus carpio against
the pathogen studied for 20 days at 5-day intervals showed increase in WBC counts.
This result is also supported by another study by Prit Benny et al.,(2010)[28]
that the musa
acuminate peel extract injected into Clarias batrachus, showed increase in the level of WBC
and lymphocytes.
CONCLUSION
The results obtained in the present study indicate that aqueous extract of leaf Annona
squamosa Linn. is a potent immunostimulant, stimulating nonspecific immune mechanisms.
The present study was evaluated that the immunomodulatory activity of aqueous leaf extract
of Annona squamosa on fish Clarias batrachus in haematological parameters exhibited
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significant increase at (10ml) 50% and (15ml) 100% concentrations of crude extracts in 30
days and post-challenged with Aeromonas hydrophila and again haematological parameters
was analysed after 15 days of treatments in the blood of the fish and which may be
considered as a sign of improvement in specific immune responses in the blood of the Clarias
batrachus Linn.
The present study shows that aqueous leaf extract of Annona squamosa has
immunostimulatory effects in Catfish with no adverse effects observed. This concludes that,
these can help in formulation of drugs.
ACKNOWLEDGEMENTS
Authors are thankful to Principal, St. Aloysius (autonomous) College, Jabalpur for lab
facilities and also thanks UGC, CRO, Bhopal for financial assistance.(vide letter No.F.MS-
80/106054/XII/14-15/CRO dated 03/03/2015).
CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest.
REFERENCES
1. Chakrabarty, N.M. (1998). Biology, in culture and production of Indian major carps –
Review (Narendra Publishing House, New Delhi, India).
2. Pandey, Govind, Sharma, Madhuri and Mandloi, A.K. Immunostimulant effect of
medicinal plant on fish. International research journal of pharmacy, 2012; 3(3): 112-114.
3. Naylor, R and Bruke, M. Ann. Rev. Environ Resource, 2005; 30: 185-218.
4. Barker, G. Bacterial diseases. BSAVA manual of ornamental fish. Wildgoose WH (Ed),
2001; 185-194.
5. Rahman, M.M., Wahed, M.I.I., Biswas, M.H., Sadik, G.M. and Haque, M.E. Science,
2001; 1: 214-216.
6. FAO. (2002). Antibiotics residue in aquaculture products. The State of world fisheries
and aquaculture 2002, Rome, Italy, 74-82.
7. Kumari, J. and Sahoo, K. P., Dietary levamisole modulates the immune response and
disease resistance of Asian cat fish, Clarias batrachus (Linnaeus), Aquaculture Research,
2006; 37: 500-509.
8. Sakai, M. Current research status of fish immunostimulants. Aquaculture, 1999; 172:
63-92.
www.wjpr.net Vol 7, Issue 1, 2018.
1004
Runa et al. World Journal of Pharmaceutical Research
9. Abdalbasit Adam Mariod, Cheah Shiau Chuen, Putri Narrima Mohd Fauzi, Sara Elkheir,
Siddig Ibrahim Abdelwahab, ‘Antioxidant activity of different parts from Annona
squamosa and Catunaregam nilotica Methanolic extract’, ACTA Sci. Pol., Technol.
Ailment, 2012; 11(3): 249-257.
10. Pardhasaradhi BV, Reddy M, Ali AM, Kumari AL, Khar A. Antitumour activity of
Annona squamosa seed extracts is through the generation of free radicals and induction of
apoptosis. Indian Journal of Biochemistry & Biophysics, August 2004; 41: 167-172.
11. Rajsekhar Saha, Pharmacognosy and pharmacology of Annona squamosa: A review.
International journal of pharmacy & life science., 2011; 2(10): 1183-1189.
12. Chitra Shenoy, M. B. Patil and Ravi Kumar. Antibacterial and Wound Healing Activity
of the Leaves of Annona squamosa Linn. Research, Journal of Pharmacognosy and
phytochemistry, 2009; 1(1).
13. Mollah MFA, Karim M. First record of induced breeding of African Magur Clarias
Gariepinus (Burchell) in Bangladesh. J. Inland. Soc, Ind., 1990; 22: 52-54.
14. Debnath, S. (2011).Clarias batrachus, the medicinal fish: An excellent c & idate for
aquaculture & employment generation. In: International Conference on Asia Agriculture
and Animal, IPCBEE, 13(2011), IACSIT, Press Singapore, 32-37.
15. Sweta Thakur and M. C. Sidhu, Phytochemical screening of leaves and seeds of Magnolia
grandiflora L., Scholars Research Library, 2013; 5: 278-282.
16. Drabkin D. R. Crystallographic and optical properties of human haemoglobin: A proposal
for the standardization of haemoglobin. Am. J. Med. Sci., 1945; 209: 268-270.
17. Blaxhall, P.C. and Daisley, K.W. Routine hematological methods for use with fish blood.
Journal of Fish Biology. 1973; 5: 771-781.
18. J.Ortuno, A.Cuesta, A.Rodriguez, M.A.Esteban and J. Meseguer. Oral administration of
yeast, Saccharonmyces cerevisiae enhances the cellular innate immune response of
giltlead sea bream (Sparus aurata). Vet. Immunol. Immunopathol, 2002; 85: 41.
19. Neethu Simon K, Santhoshkumar R and Neethu S Kumar. Phytochemical analysis and
antimicrobial activities of Annona squamosa (L) leaf extracts. Journal of Pharmacognosy
and Phytochemistry, 2016; 5(4): 128-131.
20. Samidha M. Pawaskar., K. C. Sasangan. Preliminary Phytochemical and Invitro–
Antimicrobial analysis of Annona squamosa Linn. Leaf extract. J. Pharm. Sci. & Res.,
2017; 9(5): 618-623.
www.wjpr.net Vol 7, Issue 1, 2018.
1005
Runa et al. World Journal of Pharmaceutical Research
21. Gowdhami. M, B.L. Sarkar and P.M. Ayyasamy., Screening of Phytochemicals and
Antibacterial activity Of Annona Squamosa Extracts, International Journal of
Pharmaceutical Science Invention, 2014; 3(7): 30-39.
22. A. Sivagurunathan, B. Xavier Innocent. Immunomodulatory. Effect of Dietary Cinnamon
in Growth and Haematology of Tilapia Challenged with Pseudomonas Aeruginosa Int. J.
Pharm. Phytopharmacol. Res., 2014; 3(4): 277-280.
23. A. Sivagurunathan, K. Amila Meera and Xavier Innocent. Investigation of
immunonostimulant potential of Zinger officinale and Curcuma longa in Cirrhinus
mrigala exposed Pseudomonas aeruginosa – Haematological Assessment. IJRAP, 2011;
2(3) (4): 899-904.
24. Vijayakumari Pratheepa, Nataraja Pillai and Sukumaran. Effect of Euphorbia hirta plant
leaf extract on immunostimulant response of Aeromonas hydrophila infected Cyprinus
carpio. Peerj. 671, 2014.
25. Mohsen Abdel-Tawwab, Mohammad H. Ahmad, et al. Use of Green Tea, Camellia
sinensis L, in Practical Diet for growth and production of Nile Tilapia, Oreochromis
niloticus (L) against Aeromonas hydrophila Infection. J. of the World Aquaculture
Society, 2010; 203-213.
26. T.Annalakshmi, K.M. Syed Ali Fathima B. Xavier Innocent and A.Sivagurunathan.
Evaluation of immunostimulatory potential of Phyllanthus aramus in Labeo rohita
infected with Aeromonas hydrophila: Haematological Assessment. IJRAP, Jan–Feb 2013;
4(1).
27. Pratheepa V, Ramesh S and Sukumaran N. Immunomodulatory effect of Aegle marmelos
leaf extract on freshwater fish Cyprinu s carpio infected by bacterial pathogen Aeromonas
hydrophila. Pharm Biol., 2010 Nov; 48(11): 1224-39.
28. Prit Benny, Geetha Viswanathan, Sunitha Thomas and Aruna Nair. Investigation of
Immunostimulatory behaviour of Musa acuminate peel extracts in Clarias batrachus. The
IIOAB Journal, 2010; 1(2): 39-43.