Enhancement of nonspecific immunity and disease resistance in Oreochromis mossambicus by Solanum trilobatum leaf fractions

Fish & Shellfish Immunology 23 (2007) 249e259 www.elsevier.com/locate/fsi

Enhancement of nonspecific immunity and disease resistance in Oreochromis mossambicus by Solanum trilobatum leaf fractions M. Divyagnaneswari, D. Christybapita, R. Dinakaran Michael* Centre for Fish Immunology, P.G. Department of Zoology and Biotechnology, Lady Doak College, Madurai, Tamil Nadu, India Received 16 August 2006; revised 14 September 2006; accepted 29 September 2006 Available online 11 October 2006

Abstract The purpose of this study was to determine the effect of water and hexane soluble fractions of the Indian medicinal plant, Solanum trilobatum on the nonspecific immune mechanisms and disease resistance in Oreochromis mossambicus. Fish were intraperitoneally injected with different doses of 0, 4, 40 or 400 mg kg1 body weight of water or hexane soluble fraction. The nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, reactive oxygen species production and reactive nitrogen species production by peripheral blood leucocytes. The functional immunity in terms of percentage mortality and Relative Percent Survival (RPS) was assessed by a challenge with live Aeromonas hydrophila. Almost all the doses of both water and hexane soluble fractions enhanced the serum lysozyme activity. All the doses of water soluble fraction significantly enhanced the ROS production on most of the days tested. In hexane soluble fraction treated groups, the enhancement in the ROS production was observed at least on 2 days. All the doses of water soluble fraction significantly enhanced the production of RNS only on one day. The RNS production was enhanced significantly only in the group treated with 40 mg kg1 of hexane fraction. The leaf fraction administration preceding the challenge with live A. hydrophila, decreased the percentage mortality in the experimental group with the consequent increase in RPS values. This preliminary study indicates that S. trilobatum could be used to promote the health status of fish in intensive finfish aquaculture. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Immunostimulant; Nonspecific immunity; ROS; RNS; Serum lysozyme; Disease resistance; Oreochromis mossambicus; Solanum trilobatum

1. Introduction Fish, like other vertebrates, respond to infectious agents in both nonspecific and specific ways, although they depend to a much greater extent on the nonspecific mechanisms. Immunostimulants have the ability to increase resistance to disease by enhancing nonspecific and specific defence mechanisms [1e4] and offers a promising alternative to antibiotics and vaccines [5]. The immunostimulants, which have been tested for application to aquaculture, include peptides like FK-565 [6,7], glucan [8], extracts from a tunicate [9], or an abalone [10], lactoferrin [11], levamisole [12], chitosan [13], GH [14], * Corresponding author. Tel.: þ91 452 2530527; fax: þ91 452 2521333. E-mail address: [email protected] (R.D. Michael). 1050-4648/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2006.09.015

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vitamin C [15,16], oligonucleotides from yeast RNA [17] and plant derived immunostimulants [1e4,18e20]. These immunostimulants may directly initiate activation of the innate defence mechanisms acting on receptors and triggering intracellular gene activation that may result in production of antimicrobial molecules [21]. It leads to an increase in various components of immunity such as phagocytic activity, complement activities, lysozyme and disease resistance as well as serum Ig levels. These agents are widely used to improve the impaired immune functions [22,23] and to stabilize the improved immune status. Among these immunostimulants, natural immunostimulants are biocompatible, biodegradable, cost effective and safe for the environment [24]. India is endowed with rich wealth of medicinal plants. A large number of plants have been used in traditional medicine for the treatment and control of several diseases [25]. Solanum trilobatum Linn. (Family: Solanaceae) is used in the South Indian Siddha system of medicine as an expectorant and in the treatment of respiratory diseases, asthma, chronic febrile infections, tuberculosis, cardiac and liver diseases. Sobatum, b-solamarine, solaine, solasodine, glycoalkaloid, diosogenin and tomatidine are the constituents isolated from this plant [26]. This plant possesses a broad spectrum of antibiotic, antibacterial, and anticancer activity. Many researchers have reported the medicinal importance of this plant [27e29]. Although this plant is being widely used in human medicine in India but there is no report on its application in preventing fish diseases. Hence, probably for the first time, an investigation aimed to study the effect of water and hexane soluble fractions of S. trilobatum leaves on the nonspecific immune mechanisms and disease resistance in Oreochromis mossambicus was performed. 2. Materials and methods 2.1. Fish and their maintenance Oreochromis mossambicus, a common fresh and brackish water cichlid fish was used in this study. Male fish weighing 25  5 g were used for lysozyme and disease resistance studies, and for ROS and RNS analysis, 50  5 g fish were used. All fish were maintained in Fiber Reinforced Plastic (FRP) tanks (vol. 150 l). They were kept at the ambient, uncontrolled temperature of 28  2  C under natural photoperiod. Water was changed on alternate days. Fish were acclimated for a period of 2 weeks and fed ad libitum with a balanced fish diet prepared in the laboratory [30]. 2.2. Plant extract preparation The plant, S. trilobatum were procured from the market and the plant species was identified and confirmed by Dr. D. Stephen, P.G. Department of Botany, The American College, Madurai, Tamil Nadu, India. The voucher specimen (Specimen No: CFIS01) was deposited in the Herbarium of Department of Botany, Lady Doak College, Madurai. The leaves were collected and washed in sterile distilled water. They were shade dried, powdered and stored at 20  C until further use. The extraction was done by following the methods of Lee et al. [31] and Xu et al. [32]. Ten grams of leaf powder was exhaustively extracted with methanol and it was filtered through sterile muslin cloth and the filtrate was allowed to stand for 30 min at room temperature. The filtrate was collected and the solvent was evaporated using rotary vacuum evaporator (Buchi SMP, Switzerland). The residue obtained after evaporation was mixed with sterile distilled water. The extract was taken in a separating funnel and equal volume of hexane was added and mixed carefully by intermittent releasing of the pressure inside the separating funnel. The content was allowed to stand without any disturbance till two distinct layers (lower water soluble and upper hexane soluble fraction) were seen. The fractions were collected separately. This process was repeated till the colourless hexane fraction was obtained which indicates the completion of hexane fractionation. The water and hexane soluble fractions were concentrated in rotary vacuum evaporator. The desired doses of water and hexane soluble fractions were prepared in distilled water and pure coconut oil (in order to dissolve the non-polar compounds) respectively and stored at 20  C until used for experimentation. 2.3. Experimental design To study the nonspecific immune mechanisms, fish (n ¼ 6) were injected intraperitoneally with 0, 4, 40 or 400 mg kg1 body weight of water or hexane soluble fraction using 1-ml tuberculin syringe with 24-gauge needle

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on day 0. The corresponding control fish received 0.2 ml of distilled water or purified coconut oil. The fish were bled 2 days prior to and 2, 4, 6, 8 and 10 days after treatment. 2.4. Bleeding and serum collection Fish were bled from common cardinal vein using 1 ml tuberculin syringe fitted with 24-gauge needle [33]. For serum separation, 200 ml of blood was drawn and the whole bleeding procedure was completed within 1 min. The blood was collected in serological tubes and the clot was stored in a refrigerator overnight. The clot was then spun down at 400 g for 10 min. The serum collected was stored in sterile Eppendorf tubes at 20  C until used for assays. 2.5. Lysozyme activity Lysozyme activity was measured by the method of Parry et al. [34] in combination with the microplate adaptation of Hutchinson and Manning [35]. In this turbidimetric assay, 0.03% lyophilized Micrococcus lysodeikticus in 0.05 mM sodium phosphate buffer (pH 6.2) was used as substrate. Ten microlitres of fish serum was added to 250 ml of bacterial suspension in a ‘‘U’’ bottom microtitre plate and the reduction in absorbance at 490 nm was determined after 0.5 and 4.5 min incubation at 22  C using a microplate reader (Biorad, USA). One unit of lysozyme activity was defined as a reduction in absorbance of 0.001 per min. 2.6. Preparation of viable leucocytes from peripheral blood For the separation of peripheral blood leucocytes, the fish were bled using 5-ml syringe filled with 2 ml of blood collecting medium (RPMI-1640 supplemented with 50 000 IU l1 sodium heparin, 1 00 000 IU l1 penicillin, and 100 mg l1 streptomycin). The diluted blood was carefully overlaid onto equal volume of lymphocyte separation medium (Lymphosep, ICN Biomedicals, Inc., USA) and the cells were spun down at 800 g for 20 min. The leucocytes in the interface were collected and washed twice with wash medium (RPMI-1640 supplemented with 10 000 IU l1 sodium heparin, 1 00 000 IU l1 penicillin and 100 mg l1 streptomycin) and resuspended in culture medium (RPMI1640 supplemented with 3% (v/v) of pooled tilapia serum, 1 00 000 IU l1 penicillin, 100 mg l1 streptomycin and 4 mM L-glutamine, Biochrom AG, Germany). Numbers of viable cells was enumerated using the trypan blue exclusion method and adjusted to 4  107 ml1 using culture medium. 2.7. Intracellular reactive oxygen species production The intracellular respiratory burst activity was measured by following the method of Secombes [36], with minor modifications. Peripheral blood leucocytes (1  106 cells per well) were incubated with 25 ml of nitroblue tetrazolium (NBT, 1 g l1) in 175 ml culture medium for 2 h at 28  C. The supernatants were removed and the cells were fixed with 100% (v/v) methanol for 5 min. Each well was washed twice with 125 ml of 70% (v/v) methanol. The fixed cells were allowed to air dry. The reduced NBT (in the form of formazan) was dissolved using 125 ml of 2 N potassium hydroxide (KOH) and 150 ml of dimethyl sulphoxide (DMSO) per well. Optical density was measured spectrophotometrically at 650 nm. 2.8. Reactive nitrogen species production Nitric oxide (NO) release by peripheral blood leucocytes in the medium was measured using Griess reagent. The nitric oxide produced by the cells in cultures is rapidly transformed to more stable nitrite. The nitrite present in the culture supernatant can be measured colorimetrically by converting nitrite into a pink dye by adding Griess reagent. [37]. Peripheral blood leucocytes were cultured for 96 h and 50 ml culture supernatant was collected and transferred to a separate microtitre plate. To each well containing the culture supernatant, 50 ml of Griess reagent (1% sulphanilamide, 0.1% N-naphthyl-ethylenediamine and 2.5% phosphoric acid) was added. After 10 min of incubation, the optical density was recorded spectrophotometrically at 570 nm. Molar concentrations of NO 2 were read from a standard curve generated from a graded series of NaNO2 concentrations in culture medium.

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2.9. Disease resistance Aeromonas hydrophila (AHO21, isolated from diseased freshwater fish by School of Biological Sciences, Bharathidasan University, Tiruchirappalli, India) was inoculated in tryptone soya broth and was incubated at 28  C. The culture was centrifuged at 800  g for 15 min. The packed cells were washed and the required dose was prepared in phosphate buffered saline. Groups of 10 fish each in triplicates were administered with 0.2 ml of 0, 4, 40 or 400 mg kg1 body weight of water or hexane soluble fraction of S. trilobatum either once (day 1) or twice (day 1 and 4). The control fish received 0.2 ml of distilled water or purified coconut oil. An untreated and a phosphate buffered saline injected control were also maintained. After the administration of plant extracts fish were challenged (injected) with virulent A. hydrophila (1  108 cells/fish) on day 7. Earlier the challenge dose was standardized to give 80% mortality in the untreated groups. Mortality was recorded for 15 days. The symptoms observed include, hemorrhagic septicemia, distended abdomen and lesions on the ventral surface of the body. The cause of death was confirmed by re-isolating the organism from liver of 10% of dead fish using Aeromonas isolation medium (Hi media, Mumbai, India). RPS was calculated by the following formula [38],  RPS ¼ 1 

 Percent mortality in treated group  100 Percent mortality in control group

2.10. Statistical analysis The data were expressed as arithmetic mean  standard error (SE). Statistical analysis of data involved one-way analysis of variance (ANOVA) followed by Tukey’s pairwise comparison test. The levels of significance were expressed as P-value less or greater than 0.05. 3. Results 3.1. Lysozyme activity The lysozyme activity was significantly enhanced by the administration of 40 mg kg1 of water soluble fraction (WSF) on day 4, 6 and 8 (P < 0.05). Significant enhancement was found to be on day 2 and 4 (P < 0.05) for 4 mg kg1 treated groups. In groups treated with 400 mg kg1 of WSF, significant enhancement was found to be only on one day (P < 0.05) (Fig. 1a). Lysozyme activity was also significantly enhanced in the groups treated with 4 or 400 mg kg1 of hexane soluble fraction (HSF) on most of the days tested. (P < 0.05) (Fig. 1b). 3.2. Intracellular reactive oxygen species production All the doses of WSF significantly (P < 0.05) enhanced the ROS production on most of the days tested (Fig. 2a). In HSF treated groups, fish administered with lowest dose i.e., 4 mg kg1 caused significant (P < 0.05) enhancement in the ROS production on day 2 and 4. The other doses significantly enhanced the production of ROS on day 4 and 6 (Fig. 2b). 3.3. Reactive nitrogen species production The dose of 400 mg kg1 of WSF significantly (P < 0.05) enhanced RNS production on day 6 and 8 (Fig. 3a). In 40 mg kg1 treated group, the RNS production was significantly enhanced only on day 6. There was no modulation in the group treated with the lowest dose of 4 mg kg1 of WSF. Significant enhancement (P < 0.05) of RNS production was observed in the group treated with 40 mg kg1 of HSF on day 4 and 6, and in 4 mg kg1 of HSF treated group, it was only on day 8 (Fig. 3b).

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Serum lysozyme activity (Units/ml)

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Days post treatment Fig. 1. (a) Effect of water soluble fraction (WSF) of S. trilobatum on the serum lysozyme activity in O. mossambicus. Each point represents the arithmetic mean value of six fish  standard error (*P < 0.05). (b) Effect of hexane soluble fraction (HSF) of S. trilobatum on the serum lysozyme activity in O. mossambicus. Each point represents the arithmetic mean value of six fish  standard error (*P < 0.05).

3.4. Disease resistance The percentage mortality was significantly decreased (Fig. 4a, b) in the treated fish when challenged with live A. hydrophila. Among the groups administered with single dose of plant extracts, the highest dose of 400 mg kg1 of WSF has given the maximum protection with a percentage mortality of 23.33% and RPS value of 70.84, followed by 23.33% mortality and RPS of 65.01 in group treated with 4 mg kg1 of HSF (Table 1). When administered twice, the percentage mortality of 16.67% and the maximal RPS value of 70.59 were observed in the fish administered with 4 mg kg1 of HSF. When administered twice, all the doses of HSF gave better protection compared to those of the corresponding doses of WSF.

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Days post treatment Fig. 2. (a) Effect of water soluble fraction (WSF) of S. trilobatum on the reactive oxygen species production by peripheral blood leucocytes in O. mossambicus. Each point represents the arithmetic mean value of six fish  standard error (*P < 0.05). (b) Effect of hexane soluble fraction (HSF) of S. trilobatum on the reactive oxygen species production by peripheral blood leucocytes in O. mossambicus. Each point represents the arithmetic mean value of six fish  standard error (*P < 0.05).

4. Discussion In the present study, both the fractions of S. trilobatum leaves were found to have significant stimulatory effects on the nonspecific immune mechanisms tested and disease resistance in O. mossambicus. Lysozyme is an important component in the immune system of fish. It is bactericidal by hydrolyzing b (1 / 4) linkages of bacterial cell wall peptidoglycans resulting in bacteriolysis. It is also known to act as opsonin and activate the complement system and phagocytes [39]. In the present study, it was observed that the lysozyme activity was well enhanced on treatment with water or hexane soluble fractions of S. trilobatum. Similar results of elevated lysozyme activity was observed on 20, 25 and 30 days after feeding Jian carp [40] and large yellow croaker, Pseudosciaena crocea [41] with Traditional

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Days post treatment Fig. 3. (a) Effect of water soluble fraction (WSF) of S. trilobatum on the reactive nitrogen species production by peripheral blood leucocytes in O. mossambicus. Each point represents the arithmetic mean value of six fish  standard error (*P < 0.05). (b) Effect of hexane soluble fraction (HSF) of S. trilobatum on the reactive nitrogen species production by peripheral blood leucocytes in O. mossambicus. Each point represents the arithmetic mean value of six fish  standard error (*P < 0.05).

Chinese medicine (TCM) formulated from Astragalus root (Radix astragalin seu heydsari) and Chinese Angelica root (Radix angelicae sinensis) at a ratio 5:1 (w/w). Oreochromis niloticus fed with 0.1 and 0.5% Astragalus radix root for 1 week [42] and Labeo rohita fed with 0.5% of Achyranthes aspera seed were shown to enhance lysozyme activity [43]. The activity of serum lysozyme increased significantly in Oncorhynchus mykiss fed with a marine algae, Dunaliella salina at 100 and 200 mg kg1 for 9 weeks. [44]. Respiratory burst, the increase of oxidation level in phagocytes stimulated by foreign agents, is considered as an important indicator of nonspecific defence in fish [45], where O 2 is the first product to be released [31]. Production of ROS or respiratory burst activity and RNS or NO production by phagocytes is a crucial effector mechanism for limiting the growth of fish pathogens. [46]. In recent years, NO has also been shown to be a very important molecule in regulating immune functions as well as having a direct antimicrobial effect [47]. The measurement of O 2

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Fig. 4. (a) Effect of S. trilobatum leaf fractions administered as single dose on the percentage mortality in O. mossambicus challenged with live A. hydrophila. Each point represents the arithmetic mean value of 10 fish  standard error (*P < 0.05). (b) Effect of S. trilobatum leaf fractions administered as double dose on the percentage mortality in O. mossambicus challenged with live A. hydrophila. Each point represents the arithmetic mean value of 10 fish  standard error (*P < 0.05).

concentration has been accepted as an accurate parameter to quantify the intensity of a respiratory burst. In the present investigation, all the doses of water soluble and the doses of 4 and 40 mg kg1 of hexane soluble fraction enhanced the production of ROS and RNS by peripheral blood leucocytes. This stimulatory effect may be due to the proliferative responses of leucocytes as reported by Jang et al. in rainbow trout macrophages following in vitro treatment with glycyrrhizine isolated from Glycyrrhiza glabra [18] or due to the enhancement in the degree of phagocytosis, respiratory burst activity and expression of interleukins as in the case of Ergosan injected fish [48]. The similar result of enhanced superoxide anion production by blood leucocytes was also found in the fish L. rohita fed with 0.5% Achyranthes incorporated diet [43]. However, rainbow trout fed with ginger (Zingiber officinale) extract had significantly induced the extracellular activity of phagocytic cells in blood, but not enhanced the intracellular respiratory burst activity [49].

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Table 1 Effect of S. trilobatum leaf fractions on the Relative Percent Survival (RPS) in O. mossambicus challenged with live A. hydrophila S. No.

1 2 3 4 5 6

Fractions

Relative Percent Survival (RPS) 1

WSF e 4 mg kg WSF e 40 mg kg1 WSF e 400 mg kg1 HSF e 4 mg kg1 HSF e 40 mg kg1 HSF e 400 mg kg1

Single dose

Double dose

62.5 58.33 70.84 65.01 55 55

13.04 26.08 17.39 70.59 52.94 64.70

For testing efficacy, it is very essential to estimate the increased protection in fish treated with immunostimulants [17]. The enhancement of nonspecific immune parameters by S. trilobatum leaf preparation is possibly an important factor in reducing the percentage mortality and thereby protecting the fish against live A. hydrophila challenge. Earlier studies in this laboratory also revealed that dietary supplementation of Ocimum sanctum [1] and Nyctanthes arbortristis (unpublished data) leaves and intraperitoneal injection of water soluble and hexane soluble fraction of Eclipta alba and Nyctanthes arbortristis leaves (unpublished data) enhanced the disease resistance against A. hydrophila in O. mossambicus. The present finding is in agreement with the results of Abutbul et al. [50] in tilapia fed with a diet containing ethyl acetate extract of Rosmarinus officinalis leaf powder and Fujiki et al. in carp administered with the fraction II of Undaria pinnatifida. They also found that the administration of this fraction 6 and 3 days prior to intraperitoneal challenge with E. tarda significantly increased the survival rate [51]. The disease resistance against A. hydrophila was enhanced in L. rohita fed with 0.5% of Achyranthes [43]. The methanolic herbal extracts (S. trilobatum, Andrographis paniculata and Psoralea corylifolia) helped to increase the survival and growth and reduced the bacterial load even in the shrimp, Penaeus monodon post larvae [52]. Even though, both the fractions enhanced the nonspecific immune mechanisms, the hexane soluble fraction was more protective than the water soluble fraction when it was administered twice. This enhancement in nonspecific immune responses and the protection by hexane soluble fraction might be due to the presence of the compound, Sobatum. Sobatum, a partially purified component of S. trilobatum was obtained from the petroleum ether/ethyl acetate (75:25) extractable portion, which was identified as b-sitosterol by comparison with an authentic sample and proved to be an anticancer agent by in vitro and in vivo experiments [27]. This phytosterol has shown to be effective in stimulating various components of the immune system at mammalian level [53]. In conclusion, the administration of water or hexane soluble fraction enhanced some of the nonspecific immune parameters and disease resistance against A. hydrophila in tilapia. But the hexane soluble fraction seems to be a better immunostimulant, which can have a promising role in aquaculture to prevent diseases and disease outbreaks. Also further investigation on the immunostimulatory effect of this plant preparations when administered along with feed (which is the preferred route of administration in the field) for disease prevention in aquaculture is warranted. Acknowledgments The financial support from Department of Biotechnology, Government of India, New Delhi for the study and the technical guidance for the plant extract preparation by Dr. S. Premsingh, Department of Chemistry, The American College, Madurai are greatly acknowledged. References [1] Logambal SM, Venkatalakshmi S, Michael RD. Immunostimulatory effect of leaf extract of Ocimum sanctum Linn. in Oreochromis mossambicus (Peters). Hydrobiologia 2000;430:113e20. [2] Venkatalakshmi S, Michael RD. Immunostimulation by leaf extract of Ocimum sanctum Linn. in Oreochromis mossambicus (Peters). J Aqua Trop 2001;16:1e10. [3] Logambal SM, Michael RD. Azadirachtin e an immunostimulant for Oreochromis mossambicus (Peters). J Aqua Trop 2001;16:339e47. [4] Chakrabarti R, Rao YV. Achyranthes aspera stimulates the immunity and enhances the antigen clearance in Catla catla. Int Immunopharmacol 2006;6:782e90. [5] Anderson DP. Immunostimulants, adjuvants and vaccine carriers in fish; application to aquaculture. Annu Rev Fish Dis 1992;2:281e307.

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