on clarias batrachus fish infected with aeromonas ... · the total erythrocyte count and total...

www.wjpr.net Vol 7, Issue 1, 2018.

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Runa et al. World Journal of Pharmaceutical Research

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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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

https://www.ncbi.nlm.nih.gov/pubmed/?term=Pratheepa%20V%5BAuthor%5D&cauthor=true&cauthor_uid=20818950


1003


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.

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on clarias batrachus fish infected with aeromonas ... · the total erythrocyte count and total...

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