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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407

Research Article

SPECIFIC AND NON-SPECIFIC IMMUNE RESPONSE AND DISEASE RESISTANCE OF SOLANUM TORVUM LEAF SOLUBLE FRACTIONS IN FRESHWATER CARP

CYPRINUS CARPIO V.Uthayakumar1*, V. Ramasubramanian1, D. Senthilkumar2, P.R. Sreedevi1 and S. Munirasu1 1Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore-641046 Tamilnadu, India

2Deparment of Zoology, Kandasami Kandar’s College, Paramathy Velur- 638182, Namakkal, Tamil Nadu, India

Article Received on: 08/03/12 Revised on: 11/05/12 Approved for publication: 20/05/12 *Email: abiotechuthay@gmail.com ABSTRACT The immunostumulatory effect of the leaves extract of a medicinal plant Solanum torvum (Turkey Berry), On Specific, non-specific immune responses and disease resistance of Common carp Cyprinus carpio was investigated. Fish were intraperitoneally injected with 0, .8, 80 or 800 mg kg-1 body weight, of the water soluble fraction. The non-specific humoral (Lysozyme activity) and cellular (Hematology and Agglutination) responses and disease resistance against Aeromonas hydrophila and nutraceutical effect of S. torvum leaf powder supplementation on fish Growth estimation. All the doses of water-soluble fraction tested, significantly enhanced the serum Lysozyme activities and Agglutination and Total Serum Protein on 45 days experimental Period. The highest Lysozyme activity and agglutination was observed in the group fed with 80 mg of S. trovum feed for 45 days the results were Significantly (p<0.05). Hematological parameters of experimental groups were significantly increase compared with control groups. The maximum growth was observed in 80mg S. torvum fed fish and decreased growth was observed in 0.8 mg and 800mg of herbal diet fed fish (P < 0.05) compared with non-supplemented control fish. The results clearly indicate the immunostimulatory and disease resistance properties of S. torvum leaf fraction and so its potential to be used as an first grade immunostimulants in fresh water aquaculture. KeyWords: Solanum torvum, Immunostimulant, Lysozyme activity, Agglutination. INTRODUCTION Aquaculture is the fastest growing food-producing sector in the world. The increased intensification of aquaculture has led to a high number of disease outbreaks with an increasing range of pathogens causing them (Kirubakaran et al., 2010). Immunostimulants are natural or synthetic substances that are able to activate non-specific and specific immune responses (Anderson, 1992). The use of immunostimulants for the prevention of fish diseases progresses and several preparations and regimes has become more promising (Jeney and Jeney, 2002). Herbal drugs are known to possess immune-modulatory properties and generally act by stimulating both specific and non-specific immunities. One of the most promising methods of controlling diseases in aquaculture is strengthening the defense mechanisms of fish through prophylactic administration of immunostimulants (Robertsen,1999). Substances obtained from natural sources being biodegradable and biocompatible have received attention with regard to fish. A number of plant material/products such as saponin (Ninomiya et al., 1995), glycyrrhizin (Jang et al.,1995), aloe (Kim et al.,1999), Ocimum sanctum (Logambal et al.,2000), Azadirachtin (Logambal et al., 2001), Viscum album, Urtica dioica and Zingiber of ficinale (Dugenci et al., 2003), Radix astragalin seu Hedysari and Radix angelicae sinensis (Jian, et al., 2004), Astragalus radix and Scutellari radix (Yin et al.,2006), and Achyranthes aspera (Rao et al., 2006) have been reported to enhance the immunity of fish. Solanum torvum Swartz (family: Solanaceae) commonly known as turkey berry. This plant is found in tropical Africa, Asia and South America. The fruits have been found to contain Its leaves have been used in the treatment of abdominal pain, whitlow and whooping cough; its fruits are used in the treatment of anemia, inducing lactation, and treatment of wounds and snakebite(Accra et al., 2007 and

Koffuor et al., 2011). To date there is little scientific evidence to support the traditional use of S. torvum in the management of anemia and immunodeficiency and the possible mechanisms involved. With these findings in view, the aim of this work was to evaluate the effects of dietary and intra peritoneal administration of aqueous leaf extract on both the specific and non-specific immune responses and disease resistance of C. carpio against A. hydrophila during a 45th study. MATERIALS AND METHODS Fish Collection and Maintaining C. carpio, a freshwater carp was used for the present study. Carps fries were collected from Bhavanisagar, Government Fisheries Farm, Bhavani Sagar Dam, Erode, Tamil Nadu, India, weighing 2± 2g for using growth studies, 10 ± 2g (for non-specific humoral assays and disease resistance studies) and 20± 5g (for non-specific cellular assays). Fishes were acclimated for 15 days in 500 L cement tanks. They were kept at the ambient, uncontrolled temperature of 28±2 ºC under natural photoperiod and maintain the water quality during this study. Fishes were fed Commercial feed. Preparation of extract S. torvum (Turkey Berry), leaves were collected from the Bhavani area, Coimbatore Tamil Nadu India. The leaves were dried in shade place then make powder form. Powder was stored at 20˚c by following the method of Lee et al. (2000) and xu et al. (2000). Both 100 g powder were mixed with 300ml of methanol in 500ml reagent bottle and it was kept for 3 days (72 hrs.). After 72 hours extract was filtered through sterile muslin cloth. The extract was pooled in a rotary vacuum evaporator to evaporate hexane solvent. The residue collected from the rotary vacuum evaporator was dissolved in purified coconut oil to get the desired dosage.

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Diet composition Feed ingredients viz, groundnut oil cake (32.00%), rice bran (26.10%), soybean meal (15.90%), wheat flour (20.05%), fish meal (3.95%) and Vitamins and minerals (2.0%) were used as normal diet for control. All the ingredients were properly weighed as per their inclusion rates in the four experimental diets. Diet-1=Normal feed alone; Diet-2= Normal feed+ 0.8mg S. torvum powder; Diet-3= Normal feed+ 80mg S. torvum powder; Diet-4= Normal feed+ 800mg S. torvum powder. S. torvum leaf powder was air dried and mixed with the normal feed. Bacterial Strain and Challenge Study A virulent strain of Aeromonas hydrophila received in a lyophilized form from IMTECH laboratory (Institute of Microbial Technology, Chandigarh, India) the bacterial cultures were maintained on Tryptose Soya Agar(TSA) slants (Hi-media, Mumbai) at 5°C. In all experimental groups were challenged with 100 μl of A. hydrophila at a concentration of 1.5±0.3 x 106 CFU ml-1 in PBS as a medium. The suspension in PBS was inoculated intra-peritoneally in all specimens of all the groups by 1ml syringe on 15th day and the specimens were re-challenged on 30th and 45th day. Due care was taken to avoid any injury while challenging the experimental stock with A. hydrophila. All the challenged specimens were released back into their respective glass tanks and were observed for their responses against the injected bacterial strain. Percent mortality in treated group RPS = 1- {-------------------------------------------} x 100 Percent mortality in control group Experimental Design To study the non-specific immune mechanisms, fish (n = 6) were injected intraperitoneally with 0, .8, 80 or 400 mg kg -1 body weight of water or methanol soluble fraction using 1-ml tuberculin syringe with 24-gauge needle on day 0. The corresponding control fish received 0.2 ml of distilled water or purified coconut oil. The fish were bled 3 days prior to and 3, 6, 9, 12 and 15 days after treatment. Collection of Serum from fish Fish from each treatment were anaesthetized and blood was collected 1 ml tuberculin syringe fitted with 24-gauge needle (Michael et al., 1994) and stored in 5 ml test tube without anticoagulant and allowed to clot for 2 h at room temperature in a slanting position. The tubes were centrifuged at 2500 x g for 15 min and the supernatant serum was separated and collected in screw cap sterile Eppendorf tubes at 20 ºC until used for assays. Estimation Total Serum Protein The protein content in Serum was estimated by employing Folin-Ciocalteau reagent method of Lowry et al. (1951). Haematology Blood hemoglobin was estimated by Hemoglobinometer. Erythrocyte and Leukocytes were counted by the method of Rusia and Sood, (1992) using haemocytometer. Calculation Count the RBC’S per cu.mm of the blood = (No of Erythrocyte counted X 200 X4000) / 80 Number of WBC (Thousands/cu.mm) = (No. Of leukocyte counted ×Dilution) / (Area counted depth of fluid) Serum Lysozyme Activity Serum Lysozyme activity was measured by adapting the method described by Parry et al. (1965).Serum samples were

diluted with phosphate buffer (pH 7.4) to a final concentration of 0.33 mg ml-1 protein. In a suitable cuvette, 3 ml of Micrococcus luteus (IMTECH Chandigarh, India) suspension in phosphate buffer (A450 ¼ 0.5e0.7) was taken, to which 50 ml of diluted serum sample was added. The content of the cuvette was spectrophotometer at 450 nm exactly 60 Minutes s after the addition of the serum sample. The absorbance was compared with standard lysozyme of known activity. The lysozyme activity was expressed as IU ml-1 per mg protein of serum. U/ml = (OD1-OD2/4×0.001×2) × 1000 Agglutination assay 100 µl of PBS was aliquoted into 96 well microtitre plates. The first row was used as control without the extracts. The extracts (100 µl) were added into the first well of the second row, and a 2-fold serial dilution was made until the last well in row three. Then 50 µl of bacterial pathogen was added to all the wells. They were incubated at room temperature for 1 h. The presence of buttons in the centre of the well indicates agglutination and the agglutination titre value of the extracts were read as the reciprocal of the last dilution showing agglutination. RESULTS Serum protein The results of the investigation reveal that the Total Serum protein content of C. carpio among the control and experimental groups (Figure. 1). The serum protein was increased significantly in the D3 groups for (the 45th day (P < 0.05). The highest protein content was observed in the group fed with 800 mg of S. torvum feed for days.

Fig. 1. Effect of water-soluble fraction (mg kg-1 body weight) of S.

torvum leaves on the Total Serum Protein (g/dl). Each point represents mean± SE of 6 fish (serum samples assayed in duplicate); a DMRT comparison of control and treated groups on particular days shown

significant difference (P < 0.05). Serum lyzozyme The results of the present study revealed that the Serum lysozyme activity from the serum of C. carbio was reported highest in D2 (867±0.912 be decreased in D1 (823±1.321 ±2.001 sed significantly in the fish fed for 15, 30 and 45 day with feed supplemented with different doses of powdered extract (P < 0.05). The highest lysozyme activity was observed in the group fed with 80 mg of S. trovum feed for 45 days.

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Fig.2. Effect of water-soluble fraction (mg kg-1 body weight) of S. torvum leaves on the serum lysozyme activity. Each point represents mean± SE of 6 fish (serum samples assayed in duplicate); a DMRT comparison of control and treated groups on particular days shown significant difference (P <

0.05).

Table-1. Fig.1. Effect of water-soluble fraction (mg kg-1 body weight) of S. torvum leaves on the Hematology. Each point represents mean± SE of 6 fish (serum samples assayed in duplicate); a DMRT comparison of control and treated groups on particular days shown significant difference (P < 0.05).

S.

No

Exp. Groups

Hemoglobin content in 100cc RBC (106 cmm-3) count WBC (104cmm-3) count 15th day 30th day 45th day 15th day 30th day 45th day 15th day 30th day 45th day

1 Control 6.06±0.43 07±0.26 7.04 ±0.71 1.002 x106 2.22 x106 2.32 x106 1.37x104 1.02x104 1.01x104 2 0.8 mg/kg 5.98±0.86 7.09±1.52 7.07±1.50 2.100 x106 2.34 x106 2.46 x106 1.68x104 1.65x104 1.32x104 3 80mg/kg 6.54±0.98 8.29±1.45 8.47±1.52 2.031 x106 3.02x106 3.75 x106 1.81x104 1.92x104 1.94x104 4 800mg/kg 5.64±1.09 8.37±1.61 8.08±1.43 2.020 x106 2.63 x106 2.67 x106 1.66x104 1.85x104 1.87x104

Table-1. reveals the analysis of Hematological parameters of both control and experimental groups. The hemoglobin content was high in 80mg (8.47±1.52) injected group. There is no big difference in hemoglobin level in other three groups. On the day the level of hemoglobin increased in both 80 and 800mg of S. torvum injected groups. In 800mg/kg at the end of the 45 day the hemoglobin level decreases. An increased number of RBC was recorded in 80mg/kg S. torvum injected group than 0.8 and 800mg/kg groups and control. At the end of the experimental the highest numbers of RBC were observed in 80mg/kg (3.75 x106) administrated group. Maximum WBC count was observed in 80mg/kg (1.94 X 104) and minimum observed in the control (1.01 X104).

Fig-3. Effect of water-soluble fraction (mg kg-1 body weight) of S. torvum leaves on the Growth Rate. Each point represents mean± SE of 6 fish (serum samples assayed in duplicate); a DMRT comparison of control and treated groups on particular days shown significant difference (P < 0.05). Growth rate The initial weight of the fish was 2.21±0.07 in all groups .growth of fish fed with S. torvum was weight and significantly more (P ˃ 0.05) after feeding (45 days of study) in (fig.3). The positive effect of S.torvum on growth was clearly observed in all the groups except for control fish. The maximum growth was observed in 80mg S.torvum fed fish (9.96±0.02g) and decreased growth was observed in 0.8 mg and 800mg of herbal diet fed fish (P < 0.05) compared with non-supplemented control fish.

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Table-2. Effect of water-soluble fraction (mg kg-1 body weight) of S. torvum leaves on the Agglutination Titres. Each point represents mean± SE of 6 fish (serum samples assayed in duplicate); a DMRT comparison of control and treated groups on particular days shown with different alphabets

represents significant difference (P < 0.05). Agglutination Titre

Control D1(0.8 mg) D2(80 mg) D3(800 mg)

PIR 10th Day 0.27 ± 0.0264a 03.35 ± 0.86b 05.51 ± 0.71c 07.93 ± 0.73d

SIR 45th Day 0.60 ± 0.2082a 09.32 ± 0.94b 12.19 ± 0.94 c 21.62 ± 1.15d

PIR -Primary Immune Response

SIR- Secondary Immune Response Agglutination Titre The agglutinating antibody titres was minimum on days 10 (07. 93±0.73) and reached a maximum on day 45, (21. 62±1.15) in D 3 when compared to the control group (Table-2). Although most peak titres were reached by 30 days the rate of secondary antibody production at this temperature was still notably slower than the rates of primary responses. The titres for individual fish never exceeded those of the primary response at this temperature. DISCUSSION Disease outbreaks in commercial fisheries may be controlled by the enhancement of non- specific immunity through the application of natural immunostimulants (Siwicki et al., 1994). As the alternative to chemotherapy, application of multifunctional phytochemicals in aquaculture is new venture and few works were carried out in fish (Jian and Wu, 2003). The present study revealed that intra-peritoneal administration of water soluble fraction of S.torvum leaf significantly increased the non-specific humoral and cellular responses and disease resistance against A. hydrophila. In this study, the growth rate of fish fed with herb extracts was improved when compared with the control group. The growth rate was further enhanced in the group fed the S.torvum. It is possible that this is the result of enhancement of some components of non-specific immune system of the fish, there is strong evidence that feeding glucans can modify the activity of the innate immune system of fish and increase the disease resistance in several fish species (Rao et al., 2005). Feeding carp with chitosan (1%) and levamisole (250 mg/kg of diet) also increased survival of fish following challenge with A. hydrophila (Gopalakannan and Venkatesan, 2006). The total serum protein level in the present study reveals the elevation of serum protein content in S. torvum supplement groups while, a decrease in the protein content was found in the control (fig 3). Total serum protein content at the end of experimental trial was lowest in control (infected) group and highest in D3 group. Dina Rairakhwada et al. (2007) also found that total serum protein content was significantly enhanced Similar results were obtained by Bruno and Munro, (1986), the total serum protein, were significantly (P<0.05) enhanced in levan fed common carp fingerlings against the infection of A. hydrophila while the depressed values were found in control (infected) group. Lysozyme is a fish defense element, which causes lysis of bacteria and activation of the complement system and phagocytes by acting as opsonin (Magnadottir, 2006). Lysozyme is a cationic enzyme that breaks b-1,4 glycosidic acids and N-acetyl glucosamine in the peptidoglucan of bacterial cell walls. This action is known to attack mainly

Gram positive bacteria as well as some Gram-negative bacteria in conjunction with complement. A gradual increase was found with dietary S torvum supplementation. Maximum activity was found in D3 group on 45 day (fig .2). Previous studies reported an increased protection against fish bacterial infection, which correlated with an increment in serum lysozyme levels, phagocytic activity and bactericidal activity of head kidney leucocytes. Such enhancement in lysozyme levels could also be correlated with enhanced phagocytic activity. Similar Studies on serum lyzozyme show, a significant increase in the lysozyme activities was observed in the 100, 200 and 400 mg kg-1 mixed herbal enriched diets groups on the first week did not enhanced the lysozyme activity and the second and fourth weeks, it was significantly enhanced from the control (Harikrishnan et al., 2009). Similarly elevated lysozyme activity was noted on 20, 25 and 30 days after feeding Jian carp (Chen et al., 1992) and large yellow croaker the carp spawners, with treated fish displaying elevated leucocyte and neutrophil numbers and also increase in the lysozyme levels and natural antibody titres. The supplemented feeds also enhanced the lysozyme activity in all the treatments. Cumber et al., 1981 have reported that heavy metal pollution affects the lysozyme levels causing alteration in immune-modulatory function in fish and there is a increase in the lysozyme concentration in the fish blood. Siwicki, (1987 and 1989) described the immunostimulatory activity of levamisole in of pathogens. The erythrocytes observed in Cyprinus carpio was found to be nucleated and elongated unlike the other vertebrates similar reports were well reported by (Mahajan et al., 1979) The ability of leucocytes to kill pathogenic microbes is probably one of the most important protection mechanisms. The reactive oxygen species produced by activated neutrophils and macrophages are capable of destroying the invading pathogens and are considered as an important indicator of non-specific defense in fish. Agglutinins are a group of proteins, which have different specificity for carbohydrate binding. These agglutinins are Ca2+-dependent and can agglutinate a number of fish bacterial pathogens. These probably bind to the carbohydrate moieties on the surface of bacteria, which are involved in attachment to the integumental cells, thus blocking attachment and subsequent invasion of the host. The natural agglutinin levels in serum were estimated by haemagglutination and bacterial agglutination assays. The alternate complement pathway gets activated it adheres to the agglutinating antibody Ig G, this antibody is none for memory response after the injection of the same pathogen there quicker response. Equally, the potential difference of immune responses in immature. Similar findings was

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recorded (James et al., 1987) in haemagglutinin of R. salmoninarum using rabbit erythrocyte was reported .The extraction of the haemagglutinin from R. salmoninarum at 100 "C is reminiscent of the extraction procedure used to obtain an antigen from infected tissues. This finding is in agreement with some of the earlier studies with plant-derived immunostimulants. A signifcant reduction in mortality was observed in tilapia fed with diets containing an ethyl acetate extract of Rosmarinus offinalis leaves (Abutbul et al., 2004) or an aqueous extract of E. alba leaves (Christybapita et al.,2007; Divyagnaneswari et al.2007).Brown seaweed, Undaria pinnatfida extract increased the survival rate of C. carpio against Edwardsiella tarda (Fujiki et al.,1994) and the disease resistance of L. rohita was enhanced against A. hydrophila when fed with a preparation of A. aspera (Rao et al., 2006). Recently, Ardo et al. (2008) reported that the feeds containing A. membranaceus and L. japonica extracts significantly reduced the mortality of O. niloticus followed by experimental infection with A. hydrophila. Thus, some of the active principles present in water soluble fraction are possibly, immunostimulatory in C. carpio. Based on the results of the present study it can be concluded that in C. carpio, intraperitoneal administration of water soluble fraction of S. torvum leaf, enhances the non-specific humoral and cellular immune responses and the protection against live virulent A. hydrophila as well. REFERENCES 1. Abutbul S, Goldhirsh AG, Barazani O, Zilberg D. Use of Rosmarinus

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Source of support: Nil, Conflict of interest: None Declared