Fish disease controlling efficacy study of selected Indian medicinal plant

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1 176 Original Research Article Journal Pharmaceutical, Chemical and Biological Sciences ISSN: Impact Factor (GIF): Impact Factor (SJIF): June-August 2016; 4(2): Fish disease controlling efficacy study selected Indian medicinal plant Ashokkumar R*, M. Ramaswamy Department Zoology, Karpagam University, Coimbatore 21, India *Corresponding Author: Ashokkumar R, Department Zoology, Karpagam University, Coimbatore 21, India Received: 22 October 2015 Revised: 15 July 2016 Accepted: 17 July 2016 ABSTRACT The present study is aimed to investigate the disease controlling efficacy the methanolic leaf extract Indian medicinal plant, Phyllanthus amarus, amended with manually prepared feed fed to fingerlings edible freshwater fish, Channa striatus(bloch) which was to two different concentrations (10-5 & 10-3 ) two species s, fluorescens (bacteria) and chrysogenum (fungus)in different rearing tanks. Haematological parameters control and experimental plant extract/chemical antibiotics) fingerlings revealed that the methanolic extract Phyllanthus amarushas has got higher controlling efficacy bacterial infection than fungal infection by way increasing the lymphocyte count in the blood to stimulate humoral immune response by way producing immunoglobulins to fight against harmful s. Keyword: Phyllanthus amarus; fluorescens; chrysogenum; channa striatus(bloch); lymphocyte; humoral immune response; immunoglobulins. INTRODUCTION Medicinal plants are great importance for the health individual and the society. The medicinal value these plants lies in some chemical substances that produce a definite physiological action on the human and animal body. The Indian and Chinese people depend on medicinal plants as an alternate medicine remedy against infections. Among various kinds cultivated organisms, many marine and freshwater finfish and shellfish species constitute an important industry with their production increasing every year. Recently, due to intensive farming practices, infectious diseases pose a major problem in aquaculture

2 Ashokkumar R et al 177 industry, causing heavy loss to farmers. In order to address this problem, several studies have been conducted on the modulation fish immune system in order to prevent the disease outbreak as reviewed recently by Sakai (1999) [1]. Disease outbreaks are increasingly being recognized as a potential constraint on aquaculture production and trade, and cause massive financial loss through mortality or reduced meat quality, resulting in reduced prit margins, Smith et al., 2003 [2]. The economic loss due to the diseases outbreak in the aquaculture sector can be considerable. For example, economic loss attributed due to the diseases in the Asian region countries was estimated to be at least US$ 1400 million in 1990 (ADB/NACA 1991). Antimicrobial effects different extracts various medicinal plants were studied by a number workers, Subhadradevi V et al., 2011; Anushia C et al.,2009; Ramakrishnan G et al.,2011; Kaveri Singh et al.,2010; Sathya A V et al.,2012; Mahesh B and Satish S, 2008; Foysal M J et al.,2011; Chakraborthy G S, 2008; Sathya A and Ambikapathy V, 2012; Ogunjobi A A and Ogunjobi T E, [3-12]. MATERIAL AND METHODS Collection plant Leaf sample medicinal plant species, Phyllanthus amarus was collected from Kalingarayan canal bank at Bhavani (Erode District, Tamilnadu) Identification the plant species was done with the help Dr.R.Gopalan, Pressor Botany Karpagam University (former Scientist, BSI, Coimbatore),Coimbatore. Preparation leaf extract The collected leaves were shade dried at room temperature for 20 days. The dried leaves were powdered in mechanical grinder. 20 grams leaf powder was weighed, 150ml methanol solvent was added and kept for 3 days. The extract was filtered using Whatman No.1 filter paper and the supernatant was collected. The residue was again extracted two times (with 3 days interval) and supernatants were collected. The supernatants were pooled and evaporated (at room temperature, 28± 1 C) till the volume was reduced to fully dried level. Extract the leaf powder the Phyllanthus amarus stored in air tight bottles for further analysis. Preparation and extract/chemical antibiotics-amended fish feeds. The fish feed was prepared by using known quantities ingredients such as Anchovy, Jawala (dry fish), soya flour, tapioca flour, wheat flour and rice flour to maintain the required protein level. The selected ingredients were powdered and sieved to get fine particles uniform size. Then, the ingredients were weighed according to the formulation [13] hand kneaded by adding sufficient quantity distilled water and finally made into a dough. The dough was then autoclaved in a closed aluminum container at 105 o C for about 15 minutes and then cooled. To the cooled dough, aqua savor, mono sodium phosphate, vitamin pre mix, mineral pre mix, vitamin C and vegetable oil were added and this served as the feed. The feed (Plate 13) thus prepared, was divided into five parts. One part was treated as control feed and the remaining 4 parts were used for preparing amended feeds by mixing the methanolic leaf extract P. amarus, tetracycline and fluconazole (2.5gm/50kg body weight concentrations). Preparation stock culture s Stock cultures fluorescens was whereas, chrysogenum was grown in potato dextrose agar (PDA) nutrient broth at 30 C. Sub cultures the s were also prepared and maintained at 4 C, *14+.

3 Ashokkumar R et al 178 Collection fingerlings Fingerlings Channa striatus, the freshwater, Indian murrel, vernacularly called as Verral or Snake headed fish. A bulk sample fingerlings ( 3 ± 0.5 cm length and 10 ± 2 gm Wt) Channa striatus were obtained from M/S. Murugan fish farm, Palayamkottai. The bulk samples were acclimated for 10 days in large fiber glass tanks with regular feeding with fish feed [13]. Collection blood samples for haematological analysis For haematological observations, blood sample was collected by direct cardiac puncture fingerlings using a sterile insulin needle (1ml) pre-rinsed with anticoagulant (heparin) solution. 0.5ml blood sample was obtained by pooling the blood collected from 10 fingerlings for each experimental set up. Blood samples were collected from control fingerlings prefed with feed and microbe, and fingerlings prefed with amended feed and microbe- at the end 1 st, 7 th, 14 th and 21 st days. The haematological parameters such as total RBC count, haemoglobin content, total leucocyte count (TLC) and differential leucocyte count (DLC) were estimated in the blood fingerlings Channa striatus prefed with control/amended feeds and to s [13]. RESULTS Haematological parameters in microbe fingerlings Channa striatus prefed with control and amended feed The data on the different haematological parameters control and experimental plant extract/chemical antibiotics) fingerlings to two different concentrations s are presented in Tables 1 to 8. Comparative analysis percent changes (from control level) in different haematological parameters in the blood experimental fingerlings prefed with amended feed and to s are shown in Figs. 1 to 8. (a) Total erythrocyte count The total erythrocyte count in the blood control feed and fluorescens for 6 days) fingerlings at the end 21 days ranged from 3.30 ± 0.01 to 3.40 ± cells/mm 3. The total erythrocyte count the fingerlings registered elevations (ranging from 0% to 3%) from the control levels (excepting that 10-3 concentration bacteria fingerlings prefed with ) (Table 1).

4 Ashokkumar R et al 179 Table 1. Total erythrocyte count (10 6 cells/mm 3 ) in the blood control and experimental plant extracts/chemical antibiotic) fingerlings to two different concentrations Pseudomas fluorescences and chrvsogenum. F value ± ± ± ± NS ± ± ± ± NS ± 0.01 (-8) NS 3.10 ± ± 0.01 (-3)NS 3.26 ± 0.01 (-2)NS 1.25 NS ± ± ± ± S (-1) S (+6) HS (+10)HS (+10) HS ± ± ± ± S (0) S (-5) NS (-5)NS ± ± ± ± HS (+2) S (+5) S (+7)HS (+12)HS ± ± ± ± NS ± ± ± ± NS ± 0.01 (-7) NS ± 0.01 (+5) HS ± 0.0(- 13) HS ± 0.01 (+5) HS 3.09 ± ±0.01 (-15) HS 3.33 ± 0.01 (+8) HS 3.20 ± 0.01 (-4)NS 3.20±0.01 (+6)HS 2.98 ±0.01 (-11)HS 3.32±0.01 (+10)HS 3.35 ± ± 0.01 (+9) HS 3.30 ± 0.01 (-2) HS 3.43±0.01 (+11)HS 6.48 S S 3.52 S S Values are means 6 observations ± S.E. percent from control level are given in the parenthesis (b) Haemoglobin content The haemoglobin content control fingerlings from 1 st to 21 st days after microbial exposure insignificantly ranged from ± 0.06 to ± 0.05 g/100ml. Though the patterns changes in haemoglobin content showed variation up to 14 th day, after 21 st day, the haemoglobin content registered elevations (ranging from 0% to 14%) in the blood fingerlings all the experimental conditions (Table 2)

5 Ashokkumar R et al 180 Table 2. Haemoglobin (g/100ml) count in the blood control and experimental plant extracts/chemical antibiotic) fingerlings to two different concentrations fluorescens and chrysogenum. F value ± ± ± ± NS ± ± ± ± NS ± 0.06(- 1) NS ± 0.05 (0) NS ± 0.05 (0)NS ± 0.06(+1) S 4.85 S ±0.05(+10) HS ± 0.06(+8)HS ± 0.05(+10)HS ±0.04(+13) HS HS ± ± ± ± HS 0.05 (0) NS (+1) S ± ±0.03( ± ± HS (-9) HS 10) HS (+13) HS 0.03(+14)HS ± ± ± ± NS ± ± ± ± NS ± 0.05 (0) NS ± 0.05 (+3) HS ± 0.06 (0) NS ±0.08 (+4) HS ± ± 0.05(+4)HS ± 0.08(-1)NS ± 0.05(+7)HS ± 0.02 (- 1) NS ± 0.05(+11)HS ± 0.05 (+6) HS ± 0.08(+10)HS ± 0.06 (0) S ± 0.05(+12)HS ± 0.05 (+6)HS ± 0.02(+13)HS HS HS HS HS Values are means 6 observations ± S.E. Percent changes from control levels are given in the parenthesis. (c) Total leucocytes count (TLC) The total leucocytes count control fingerlings Channa striatus up to 21 days ranged between ± 0.04 to ± cells/mm 3 which were found to be statistically insignificant. However, following 21 days, fingerlings extracts and. chemical antibiotics and microbe ) registered significant elevations (over control level) in the total leucocytes count ranging from 0% to 3% excepting that fingerlings prefed with fluconazole and fungus, which showed -1% reduction from control level (Table 3).

6 Ashokkumar R et al 181 Table 3. Total leucocyte count (10 3 cells/mm 3 ) in the blood control and experimental plant extracts/chemical antibiotic) fingerlings to two different concentrations fluorescens and chrysogenum. F value ± ± ± ± HS ± ± ± ± NS ± ± ± ± HS (-4) NS (0) S ± 0.02 (-9) NS ± 0.02 (-5) NS ± ± 0.05 (0) S S ± 0.08 (-8) NS ± 0.07 (-8) NS ± 0.07 (-4) NS ± 0.05 (0) S S ±0.05( ± ± ± HS 10) NS (-9) NS (-5) NS (0) S ± ± ± ± NS ± ± ± ± NS ± ± ± 0.01 (-7) NS ± ± ±0.01 (-10) NS ± 0.05 (-5) NS 11.90± ± ± 0.01 (-5) NS ± ± ± 0.01 (+1) S ± 0.02 (+1) NS ± 0.03 (+3) HS ± 0.05 Values are means 6 observations ± S.E. Percent changes from control levels are given in the parenthesis S 6.45 S HS 8.52 S (d) Differential leucocyte count (DLC) Basophil count The basophil count in the blood control fingerlings insignificantly ranged from 3.00 ± 0.04 percent to 2.00 ± 0.02 percent following 1 st to 21 st days. Unlike previously mentioned parameters, the basophil count uniformly showed significant reduction (from control level) ranging from 0% to -67% up to 21 days under various experimental conditions. This reduction in basophil content, inspite elevated TLC level (Table 4) is noteworthy for discussion.

7 Ashokkumar R et al 182 Table 4. Basophil count (percentage) in the blood control and experimental plant extracts/chemical antibiotic) fingerlings to two different concentrations fluorescens and chrysogenum. F value ± ± ± ± NS ± ± ± ± NS 10-3 (0) NS 3.00± S 10-5 (-4) NS 4.86 S ± HS (-33)NS (-67) NS (-34) NS 10-5 (-4) NS 4.00 ± 0.01 (-30) HS (-34) NS 3.64 S ± ± ± ± NS ± ± ± ± NS 10-3 (0) NS ± 0.03 (-33)NS ± 0.04 (-35)NS 2.00 ± ± 0.03 (-34) NS 2.00 ± 0.02 (-34) NS 2.00 ± 0.05 (-34) NS 2.00 ± 0.03 (-34) NS (-34) NS 1.00 ± 0.02 (-67) NS 2.00 ± HS HS HS HS Values are means 6 observations ± S.E. Percent changes from control levels are given in the parenthesis. Neutrophil content The neutrophil count control fingerlings insignificantly ranged from ± 1.12 percent to ± 1.12 percent following 21 days experimental period. On the other hand, fingerlings prefed with leaf extract/fluconazole and to chrysogenum showed significant reduction (from control level ranging from -3% to -18%) in the neutrophil content after 21 days (Table 5).

8 Ashokkumar R et al 183 Table 5. Neutrophil (percentage) count in the blood control and experimental plant extracts/chemical antibiotic) fingerlings to two different concentrations fluorescens and chrysogenum. (prefed with (prefed with F value ± ± ± ± NS ± ± ± ± NS ± ± ± ± S (+10)S (+7)S (+15) S ± ± ± ± HS (+22)S (+19) S (+22) S (+27) HS ± ± ± ± HS (+10) S (+25) HS (+47) HS (+35) HS ± ± ± ± HS (+37) HS (+30) HS (+32) HS (+20) S ± ± ± ± S ± ± ± ± S ± 2.00 (-10) NS ± ± ± 1.11 (0) NS ± 2.01 (+9)S ± 2.00 (-15) NS ± 1.33 (-10) NS ± ± 1.13 (-8) NS ± 1.99 (-13) NS ± ± 1.08 (-5) NS ±1.53 (-18) NS ± 1.12 (-18) NS ± 1.41 (+2) S ± 1.12 (-8) NS S S 6.65 S 7.28 S Values are means 6 observations ± S.E. Percent changes from control levels are given in the parenthesis. Eosinophil count The eosinophil count control fingerlings insignificantly ranged from 2.00 ± 0.06 percent to percent following 21 days experimental period. Unlike the neutrophil count, both under fluorescens and chrysogenum exposures, the experimental fingerlings uniformly showed - 50% reductions (from control level) following 21 days under all the experimental conditions (Table 6).

9 Ashokkumar R et al 184 Table 6. Eosinophil (percentage) count in the blood control and experimental plant extracts/chemical antibiotic) fingerlings to two different concentrations fluorescens and chrysogenum. F value ± ± ± ± NS ± ± ± ± NS HS 10-5 (-52) NS 2.00 ± 0.02 (-52) NS (-52)NS S ± ± HS (0) NS (-52) NS (-52)NS HS (0) NS (0) NS (-50)NS ± ± ± ± NS ± ± ± ± NS ± ± ± 0.03 (-52) NS (-52) NS 1.00 ± ± ± HS HS HS HS Values are means 6 observations ± S.E. Percent changes from control levels are given in the parenthesis. Monocyte count The monocyte count in the blood control fingerlings Channa striatus after 21 days experimental period following microbial exposure insignificantly ranged from 4.02 ± 0.02 percent to 2.00 ± 0.02 percent (Table 7). From Table 7, it could be observed that the monocyte count in the blood experimental fingerlings showed reductions ( different magnitudes) following 1 st, 7 th, 14 th and 21 st days after microbial exposure. The reduction percentages different experimental fingerlings significantly ranged from -25% to -51% (Table 7).

10 Ashokkumar R et al 185 Table 7. Monocyte (percentage) count in the blood control and experimental plant extracts/chemical antibiotic) fingerlings to two different concentrations fluorescens and chrysogenum. F value ± ± ± ± NS ± ± ± ± NS 10-3 (-26)NS HS 10-5 (-26)NS 2.00 ± 0.02 (-50)NS 2.00 ± HS 10-3 (-75) NS 2.00 ± 0.02 (-50)NS 3.00 ± 0.02 (-26) NS S 10-5 (-75) NS (-50)NS 3.00 ± 0.02 (-25) NS S ± ± ± ± NS ± ± ± ± NS 10-3 (-26) NS ± ± 0.03 (-52)NS (-26)NS (-52)NS 2.00 ± 0.03 (-50)NS 2.00 ± ± 0.06 (-26) NS 2.00 ± ± S HS HS HS Values are means 6 observations ± S.E. Percent changes from control levels are given in the parenthesis. Lymphocyte count: The lymphocyte count in the blood control fingerlings up to 21 days after microbial exposure insignificantly ranging from ± 2.03 percent to ± 1.42 percent. Experimental fingerlings to fluorescensuniformly showed reductions (from control level) in the lymphocyte count ranging from 0% to - 47% following 1 st, 7 th, 14 th and 21 st days. On the other hand, experimental fingerlings to chrysogenum registered elevations in the lymphocyte count ranging from 0% to +22% following all the experimental periods (Table 8).

11 Ashokkumar R et al 186 Table 8. Lymphocyte (percentage) count in the blood control and experimental plant extracts/chemical antibiotic) fingerlings to two different concentrations fluorescens and chrysogenum. (prefed with (prefed with F value ± ± ± ± NS ± ± ± ± NS ± ± ± ± S (6) S (0) NS ± 1.15 (-12) NS ± 1.16 (-12)NS ± 2.00 (-12)NS ± 1.89 (-16) NS 6.42 S ± ± ± ± HS (0) NS (-12)NS (-28)NS (-22) NS ± ± ± ± HS (-24) NS ± 2.02 (-22)NS ± 2.02 (-18)NS ± 2.02 (-12) NS ± NS ± ± ± ± NS ± 2.21 (+10) S ± 1.82 (+8) S ± 2.00 (+10) S ± 1.10 (+8) S ± ± 1.13 (+12) S ± 1.21 (+14) S ± 1.43 (+10) S 58.00± 2.11 (+14)S ± 1.32 (+18) S ± 1.12 (+10) S ± 2.01 (+12) S ±1.32 (+19) S ± 1.42 (+22) HS ± 1.31 (+8) S ± 1.02 (+14) S HS HS 3.58 S 6.72 S Values are means 6 observations ± S.E. Percent changes from control levels are given in the parenthesis. DISCUSSION Among the hematological parameters, significant increases were observed in the population neutrophils and lymphocytes in the blood fingerlings to s and fed with feed amended with the methanol extract the Phyllanthus amarus medicinal plant compared to absence significant change in the above said parameters in fingerlings treated with and fed with feed amended with chemical antibiotics. This probably indicates the possible immunostimulant potential the plant extract by way stimulating humoral mediated immune response towards microbial disease control in fish. Absence significant increase in monocytes in fingerlings to s and fed with feed amended with plant extract probably indicate the absence cell mediated immune response in the fish. CONCLUSION From the results obtained, it is concluded that the haematological parameters control

12 Ashokkumar R et al 187 and experimental plant extract/chemical antibiotics) fingerlings revealed the methanolic extract Phyllanthus amarusto to controlling efficacy bacterial infection than fungal infection and the increases lymphocyte to stimulate humoral immune response by way producing immunoglobulins to fight against harmful s. CONFLICT OF INTEREST STATEMENT The authors declare that they have no conflict interests. REFERENCES 1. Sakai M. Current research status fish immunostimulants. Aquaculture 1999; 172: Smith VJ, Brown JH, Hauton C. Immunostimulation in crustaceans: does it really protect against infection. Fish Shellfish Immunol 2003; 15(1): Subhadradevi V et al. Antimicrobial activity leaves and flowers Cassia auriculata linn. Bangladesh J Scient Ind Res 2011; 46(4): Anushia C et al. Antibacterial and antioxidant activities in Cassia auriculata. Global J Pharmacol 2009; 3(3): Ogunjobi AA, Ogunjobi TE. Comparative Study Antibacterial Activities Garcinia kola and Carica papaya. African J Biomed Res 2011; 14: (2): Ramakrishnan G et al. In vitro Antibacterial activity different extracts leaves Coldenia procumbens. Int J Pharm Tech Res 2011; 3(2): Kaveri Singh et al. Study antimicrobial activity medicinal plants against various multiple drug resistance pathogens and their molecular characterization and it s bioinformatics analysis antibiotic gene from genomic database with degenerate primer prediction. Int J Biol Technol 2010; 1(2): Sathya AV et al. Studies on the phytochemistry, antimicrobial activity and antioxidant properties Cassia occidentalis L. Asian J Plant Sci Res 2012; 2 (4): Mahesh B and Satish S. Antimicrobial activity some important medicinal plant against plant and human pathogens. World J Agri Sci 2008; 4 (S): Foysal M J et al. Antibiotic sensitivity and in vitro antimicrobial activity plant extracts to fluorescens isolates collected from diseased fish. Int J Nat Sci 2011; 1(4): Chakraborthy G S. Antimicrobial activity the leaf extracts Calendula ficinalis linn. J Herbal Med Toxicol 2008; 2 (2): Sathya A, Ambikapathy V. Studies on the phytochemistry, antibacterial activity and green synthesis nanoparticles using Cassia tora L. against amphicillin resistant bacteria. Asian J Plant Sci Res 2012; 2(4): Arthi Manju, Felicitta RJ, Sakthivel M, Haniffa MA, Valliammal S, Chelladurai G. Effect water probiotics on growth performance Channa punctatus. Int J Appl Biores 2011; 1: Ashokkumar R, Ramaswamy M. Comparative study on the antimicrobial activity leaf extracts four selected Indian medicinal plants against aeruginosa, fluorescens, chrysogenum and restrictum. J Chem, Biol, Phys Sci 2013; 3(2), Cite this article as: Ashokkumar R, M. Ramaswamy. Fish disease controlling efficacy study selected Indian medicinal plant. J Pharm Chem Biol Sci 2016; 4(2):