Indian Journal of Marine Sciences Vol. 38(1), March 2009, pp. 22-27 Evaluation of immunomodulatory activity of extracts from marine animals Aditya S Akerkar 1, Chetan A Ponkshe 2 & Madhavi M Indap 1* 1Department of Zoology, D G Ruparel College, Senapati Bapat Marg, Mahim, Mumbai 400 016, India. 2Department of Zoology, Sathaye College, Dixit Road, Vile-Parle (E), Mumbai 400 057, India. [Email: [email protected]] Received 16 August 2007; revised 21 November 2007 The whole body ether extracts of a marine prawn Nematopaleamon tenuipes (PEP), two gastropods viz . Euchelus asper (EAE) and Hemifusus pugilinus (HPE), and acetone extract of a fish Rastrelliger kanagurta (MA), were tested for their effects on Delayed type Hypersensitivity (DTH) reaction and Plaque Forming Cell (PFC) assay. The Delayed type Hypersensitive reaction assay for HPE and PEP as well as MA showed stimulation but EAE was found to be less effective . In the PFC assay HPE and MA showed immunostimulation whereas PEP and EAE showed immunosuppression. PEP was further resolved into two fractions, which were tested for in vitro lymphocyte proliferation assay as well as antiproliferative assay. It is concluded that the test extracts possess immunomodulatory property. [Keywords : Animal extracts, Delayed Type Hypersensitivity, Plaque Forming Cells ] Introduction N. tenuipes, E. asper and H. pugilinus have already Majority of the species diversity is found in the been tested for their in-vitro and in-vivo phagocytic ocean fringe. This slender land sea interface with its activity 6. Rastrelliger kanagurta , locally known as high concentration of species is amongst the most Bangada, or Indian mackerel, found throughout biodiverse and productive environments on planet 1. Indian coastline is a rich source of protein and fatty The intense concentration of species coexisting in this acids. limited expanse makes them competitive and The regulation of immune response, whether to complex. This has led to development of chemical stimulate when required or to suppress when means by which defense against predation and unwanted is a major subject of research. This requires overgrowth is achieved. In view of this many marine use of immunomodulatory drugs. Immunomodulators bioactive compounds such as terpenoids, alkaloids, are materials, which modify body’s defense polyketides, peptides, shikimik acid derivatives, mechanism 7. sugars, steroids and a multitude of other secondary A number of in-vitro and in-vivo test systems are 2 metabolites have been isolated from marine animals . available for screening immunomodulatory activity. The animals Nematopaleamon tenuipes, an Delayed type Hypersensitivity (DTH) reactions are arthropod, is a source of high-grade edible protein and manifestations of T cell effector arm of the immune other organic matter. A bioactive steroid isolated from system and are used for studying preliminary 3 N. tenuipes showed haemolytic activity . Chitosan, a inflammatory processes, whereas in the Plaque compound isolated from its shell is used as an anti- Forming Cell (PFC) assay B cell function can be 4 obesity agent . Euchelus asper and Hemifusus assayed for reaction against antibody. Lymphocyte pugilinus both gastropods, are fairly common in proliferation also has an important role in rheumatic occurrence in the littoral zone. These animals have diseases. High numbers of T. lymphocytes are found not been tested for their bioactivities, except that a in peripheral blood of patients with lupus as well as in 5 potent coagulant has been derived from H. pugilinus . those patients suffering from rheumatoid or 8 ___ _________ autoimmune diseases . Therefore modulation of *Corresponding author lymphocyte proliferation is quintessential. th This paper won best presentation award at 7 Asia Pacific Marine The present study attempts, utilizing above Biotechnology conference by Wonder care Pharma to attend 8 th IMBC at Israel. Part of the paper was also presented at 8 th mentioned marine animal extracts for screening their International Marine Biotechnology Conference, Eilat, Israel. immunomodulatory potencies using PFC and DTH AKERKAR et al .:IMMUNOMODULATORY ACTIVITY OF EXTRACTS FROM MARINE ANIMALS 23 assays as well as testing for their activity on mixture of Petroleum ether: Ethyl acetate (1:1) were lymphocyte proliferation and antiproliferative effects obtained. on cancer cell line. Delayed type Hypersensitive reaction Material and Methods The method followed was adapted from Doherty, Test animals N S and modified by Kulkarni et al 10 . Animals were A prawn, N. tenuipes (Henderson), Phylum- divided into four groups of six animals each for the Arthropoda, two gastropods, Phylum- Mollusca, i.e. treatment with N. tenuipes , E. asper and H. pugilinus E. asper (Gmelin) and H. pugilinus (Born) and a fish extracts while the animals were divided into 5 groups Phylum- Chordata, Sub-Phylum- Vertebrata, Class- for testing R. kanagurta extract. One group served as Pisces, R. kanagurta (Cuvier) Indian Mackeral were control while the remaining served as experimental used. Prawns were collected from landing site at groups. Mice were sensitized by injecting 0.1 ml Versova, Mumbai and sun dried . E. asper was hand suspension of freshly obtained Sheep Red Blood Cells picked from rocky shore, while H. pugilinus were (SRBC) (1×10 8 cells/ml), intraperitoneally (i. p.) on collected from fishermen going for deep sea, at Khar- day one. Experimental groups received 0.2 ml of test danda and R. kanagurta was purchased from Sasoon extracts for eight days. On day 8, 2 hr after giving the dock. extracts, animals were challenged by injecting 0.1 ml Procedure for extraction of SRBC intradermally in left hind foot pad. The Dried N. tenuipes was weighed and powdered. The thickness of the food-pads was measured before powder was packed in Soxhlet apparatus and refluxed challenge and at 24 hr after challenge. The difference with petroleum ether (60-80 °C). The extracts were between 0 and 24 hr values of footpad thickness were dried over sodium sulphate, solvents recovered under taken as a measure of DTH reaction and the mean reduced pressure and stored at –20°C until further use. percent edema was determined. Extract was labeled as PEP. Mean final reading − Mean initial reading Live specimens of E. asper and H. pugilinus were %Edema = ×100 brought to the laboratory. They were de-shelled, Mean initial reading weighed, cut into small pieces and immersed in methanol. The cold percolation was repeated 3 times, Plaque Forming assay each time adding fresh methanol. The crude methanol The assay was carried according to Jerne-Nordin 11 . extract was solubilized using diethyl ether. Ether Animals were divided into five groups of six animals soluble fraction obtained was evaporated and stored at each. One group served as control while the –20°C till further use. The extracts thus obtained were remaining served as experimental groups. Mice were labeled as EAE and HPE respectively. intraperitoneally, injected with test extracts for 7 days. Fresh animals R. kanagurta were de-boned and On day eight, animals were injected with SRBC grinded into fine paste and immersed in acetone. The (1×10 8 cells/ml). Four days later, mice were sacrificed cold percolation was repeated twice, each time adding and their spleens processed into single cell suspension fresh acetone. The extract was concentrated under in Hank’s Balanced Salt Solution (HBSS). To 0.5 ml reduced pressure, labeled as MA and stored at –20°C of 0.5 % agarose prepared in HBSS, 50 µl of 7 % till further use. SRBC and 50 µl of spleen cell suspension were added Fractionation of PEP and the mixture is poured over glass slide. The slides After biomass extraction with an adequate solvent were allowed to solidify and incubated with fresh system, the first step in the isolation of a natural rabbit serum as a source of complement for 1 hr at compound from the main extract usually consists of a 37 °C. The plaques were counted using a hand- sequential gradient partition with solvents. The magnifying lens against a strong light source. The fractions so obtained contain compounds distributed result was interpreted in terms of number of Plaque 6 according to their polarity 9. PEP thus obtained by Forming Cells/10 splenic cells counted. Soxhlet extraction was loaded onto a silica column (60-120 mesh). Then further by sequential gradation In vitro lymphocyte proliferation assay of solvents according to polarity two fractions Lymphocyte proliferation assay was carried out as 12 F1 eluted with Petroleum ether and F 4 eluted with a described by Hansen M. et al . to test the activity of 24 INDIAN J. MAR. SCI., VOL. 38, NO. 1, MARCH 2009 PEP and its fractions (F1 & F 4) on human B (SRB) dissolved in 1% acetic acid. SRB was lymphocytes. Cells were plated in 96 well plates at a quickly removed and plate was washed four times concentration of 1.5×10 5 cells/well. Extracts were with 1% acetic acid to remove any unbound dye. The dissolved in DMSO and dilutions prepared in plates were air-dried and the bound dye was RPMI-1640 supplemented with 10% FBS. Extract at solubilized with 200 µl 10 mM Tris buffer ( pH 10.5). various concentrations was added to the wells and The absorbance was read at 540 nm using micro plate incubated for 24 hr in 5% CO 2. Control group reader. received same amount of DMSO. The proliferation of lymphocytes was noted by reduction of the yellow Statistical Analysis dye 3-(4,5-dimethyl-2-thiazo-lyl)-2,5-diphenyl-2H- Experiments were repeated thrice and results are tetrazolium bromide (MTT) to form a blue product. expressed as mean ± S D Data was analyzed by After 4 hr of incubation, the formazan product of Students’ t-test. MTT reduction was dissolved in DMSO, and absorbance was read using multiplate reader. The Results drug effect was quantified as percentage of control In vivo assays absorbance of the reduced dye at 540 nm.