RESEARCH ARTICLES CURRENT SCIENCE, VOL. 114, NO. 6, 25 MARCH 2018 1214 *For correspondence. (e-mail: drsreekumar@rediffmail.com) In vitro and in silico validation of anti-cobra venom activity and identification of lead molecules in Aegle marmelos (L.) Correa N. C. Nisha 1 , S. Sreekumar 1, *, D. A. Evans 2 and C. K. Biju 1 1 Saraswathy Thangavelu Centre, Biotechnology and Bioinformatics Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Puthenthope, Thiruvananthapuram 695 586, India 2 Department of Zoology, University College, Thiruvananthapuram 695 034, India Venomous snakebite is a global serious health issue and in India high rate of mortality is caused by Naja naja (Indian cobra). To evaluate anti-cobra venom ac- tivity and identify lead molecules in Aegle marmelos, in vitro and in silico screening was carried out. Leaves, stem and root bark of A. marmelos were extracted in ethanol, methanol and hexane and maximum yield was obtained in methanol. All extracts were used for testing in vitro anti-haemolytic, inhibition of anti- acetylcholinesterase and anti-proteolytic activities. The results revealed that ethanol extract of root bark has high anti-haemolytic activity, methanol extracts of leaves have the highest inhibitory effect on venom in- duced anti-acetylcholinesterase activity and ethanol extracts of leaves have maximum anti-proteolytic activity. Docking between 81 phytochemicals from A. marmelos and each of the 14 cobra venom toxic pro- teins revealed that the plant contains potential mole- cules for detoxification of all the cobra venom proteins. Keywords: Aegle marmelos, cobra venom, docking, phytochemicals, snakebite. THE annual global snakebite death rate is ~125,000, of which 50,000 are Indians 1,2 and 80% of the victims depend on traditional healers and such details are not properly documented. Considering the high death rate, the World Health Organisation has included snakebite in the list of neglected tropical diseases. Snake venom is a complex mixture of bioactive compounds such as enzymatic and non enzymatic proteins, peptides, lipids, nucleotides, car- bohydrates and amines. Of these, 90% of dry weight con- stitutes proteins 3 . Over 62 pharmacologically active 4 and 20–25 toxic molecules have been reported from snake venom, of which 12 toxic proteins are common, which may induce cytotoxicity, neurotoxicity, haemotoxicity, cardiotoxicity and myotoxicity 5 . The venom composition may vary from species to species, habitat and age of the snake. Due to this complexity and inconsistency, it is difficult to identify a single medicine against snakebite. Immunotherapy is the only treatment against snake envenomation in modern medicine. However, it induces serious side effects such as anaphylaxis, inflammation and immune reaction in patients. The scarcity of quality venom, its storage, inconsistency in venom composition, high cost, etc. are other constraints in immunotherapy. Since time immemorial, herbal medicines have been used against snakebites. In India over 350 plant species have been reported as antidotes to snake venom, but their effi- cacy and molecular mode of drug action have seldom been scientifically demonstrated. Among the four common venomous snakes in India, a high death rate is caused by the Indian cobra (Naja naja) 6 . Aegle marmelos is a common medicinal tree dis- tributed throughout India with its root, stem bark and leaves being used against snake envenomation 7,8 . How- ever, there is a controversy on its anti-snake venom acti- vity 9 . In these backdrops, the present study was aimed to evaluate the anti-cobra venom activity of A. marmelos through in vitro method, and identify lead molecules against each cobra venom toxic protein through in silico method. Materials and methods In vitro anti-cobra venom activity assays Preparation of plant extracts and venom sample: Leaves, stem and root bark of Aegle marmelos collected from 5 to 10-year-old field grown plants were shade dried and powdered separately. Ten gram powdered samples of each plant part was soaked separately in 100 ml hexane, ethanol and methanol for 72 h. The extracts were filtered using Whatman no. 1 filter paper and concentrated. Each extract was dried and total yield was estimated. The dried extracts were dissolved in normal saline after mixing with Tween20 and used for further experiments. A herbarium specimen of the experimental plant was deposited in the JNTBGRI herbarium with accession number TBGT30702. Lyophilized venom of Naja naja was procured from Haffkine Institute, Parel, Mumbai, and preserved at 4C (No. HITRT/ZNS-VAU/VM/43/1047).