Original Article EFFECTS OF DRUGS AGAINST ANTIOXIDANT AND CYTOTOXIC (HEp 2 CELL LINE) ACTIVITY COMPOUNDS FROM MARINE ANIMALS CONUS AMADIS VENOM (GMELIN, J.F, 1791) RAMESH S. 1 *, E. DILIPAN 1 , P. MAYAVU 1 1 Received: 25 Apr, 2014 Revised and Accepted: 29 May, 2014 Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences Annamalai University Parangipettai 608502, Tamilnadu, India. Email: rameshmarine4@gmail.com ABSTRACT Objective: The present study was carried out to explore the hemolytic, antioxidant and cytotoxic activity of conotoxins extracted from the venom of C. amadis. Methods: The crude conotoxin was extracted and tested for cytotoxic, antioxidant activity and cancer cell lines. The cytotoxic activity was studied by brine shrimp cells and in-vitro antioxidant activity were determined by DPPH, Superoxide anion radical scavenging assay and hydrogen peroxide scavenging from active fraction of conopeptides. The active fractions were tested in HEp-2 cancer cell lines at different concentration. Results: The different dose tested viz 100μg/mL -1 , 50μg/mL -1 , 25μg/mL -1 , 12.5μg/mL -1 , 6.25μg/mL -1 , and 3.2μg/mL -1 cytotoxicity on HEp-2 cell was observed at the 25μg/mL. HEp-2 cell displayed dose dependent decreased in viability detected as early as 48 hrs. The purified conotoxin was showed remarkable cytotoxicity against HEp-2 cell in a dose depend manner. The isolated conotoxin that produced maximum effect at 100μg/mL -1 and the half inhibitory concentrations (IC 50 ) was 25μg/mL -1 Conclusion: The present study revealed that cono-peptides from C. amadis have been used as an accessible source of natural antioxidants as well as anticancer after trial with animal and pre-clinical studies. in the concentration of 45.6±0.8%. Keywords: Gastropod, Conus amadis, Venom gland, Antioxidant and HEp-2 Cell line and MTT assay. INTRODUCTION Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent which reactions can produce free radicals. In this, antioxidants terminate these chain reactions by removing free radical that intermediates and inhibit other oxidation reaction [1]. Uncontrolled assembly of free radicals that outbreak the macromolecules such as membrane lipids, proteins and nucleic acids may lead to many health disorders such as cancer, diabetes, neurodegenerative and in flammatory diseases with severe tissue injuries [2,3,4]. In addition to that, it blocks the oxidation process by neutralizing the free radicals such as, superoxide anion radical (O2-) and hydroxyl radical (OH) which are unavoidable consequence in an aerobic organisms [5]. Reactive oxygen and nitrogen species (ROS/RNS) are continuously produced in the human body and they are controlled by endogenous enzymes (superoxide dismutase, glutathione peroxidase and catalase). However, if there is an over- production of these species, an exposure to external oxidant substances or failure in the defense mechanisms, damage to valuable biomolecules (nucleic acids, lipids and proteins) may occur [6]. Recent scientific studies have reported that the hydrolysis of proteins from plant and animal sources to acquire the compounds which show antioxidant activity has been productive, such examples include capelin protein [7], quinoa seed protein [8], canola [9], egg- yolk protein 10], milk casein [11], hoki frame protein [12], mackerel protein [13]. The bioactive compounds reported in these studies are peptides which are released by the enzymatic hydrolysis of proteins [14]. In this context, the focus of research in antioxidant drugs has now shifted towards antioxidants obtained from natural or organic sources. Venoms are key evolutionary innovations of several animal lineages, consisting of an arsenal of peptides and proteins, designed both immobilizing prey and as defense against predators [15]. The search for cancer cure from natural product (plant and animal) has been practiced for over a century and the use of purified chemicals to treat cancer still continues. As the infectious diseases are evolving and develops resistance to existing pharmaceuticals, the marine environment provides a novel source for the development of lead compounds against fungal, parasitic, bacterial and viral diseases. The earliest efforts in this field derived from the interests of marine biologists and naturalists who found a number of unique toxins that were present in diverse marine life. Cone snails obtain incredibly potent peptide toxins (conotoxins) to immobilize prey fish [16]. A number of these conopeptides reduce pain in animal models and several are now in preclinical and clinical development for the treatment of severe pain often associated with diseases such as cancer [17]. In order to identify a potent molecule, the present study was carried out to explore the hemolytic, antioxidant and cytotoxic activity of conotoxins extracted from the venom of C. amadis. MATERIALS AND METHODS Preparation of venom extract Live specimens of C. amadis were collected from Mudasalodai landing centre nearby Portonova in the South east coast of Tamil Nadu (11 o , 29’N; 79 o , 44’E). Snails were captured at a depth of 5–80 m using a trawl net attached to fishing gear. The collected snails were dissected and a crude extract was prepared from the venom gland as described by Saravanan et al. [18]. The crude extract mixture was centrifuged at 17,200 rpm for 10 min at 4 o C. The supernatant (considered to be crude extract) was retained and stored at -20 o Estimation of radical scavenging activity (RSA) using DPPH Assay C for further use [19]. The RSA activity of different extracts was determined using DPPH assay according to the methods of Nenadis and Tsimidou [20]. The decrease of the absorption at 517 nm of the DPPH solution after addition of the antioxidant (Venom extract) was measured in a cuvette containing 3550 µl of 0.1 ml, methanolic DPPH solution was mixed with 40 µl of 15 - 210 µg/mL of venom extract. Blank containing 0.1 ml of methanolic DPPH solution without venom extract and vortexed thoroughly, the setup was left at dark room temperature for 20 min and absorption was monitored after the incubation. The ability to scavenge DPPH radical was calculated by the following equation. International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 6, Issue 7, 2014 Innovare Academic Sciences