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In Vitro Antibacterial, Alpha-Amylase Inhibition Potential of Three Nudibranchs Extracts from South East Coast of India

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In Vitro Antibacterial, Alpha-Amylase Inhibition Potential of Three Nudibranchs Extracts from South East Coast of India Journal of Coastal Life Medicine 2013; 1(3): 186-192 186 Journal of Coastal Life Medicine journal homepage: www.jclmm.com Document heading doi:10.12980/JCLM.1.2013C1053 2013 by the Journal of Coastal Life Medicine. All rights reserved. 襃 In vitro antibacterial, alpha-amylase inhibition potential of three nudibranchs extracts from South East coast of India Giji Sadhasivam, Arumugam Muthuvel*, Wanjale Mrunal Vitthal, Abirami Pachaiyappan, Mohan Kumar, Balasubramanian Thangavel Centre of Advanced Study in Marine Biology Faculty of Marine Sciences, Annamalai University, Parangipettai-608 502, India PEER REVIEW ABSTRACT Peer reviewer Objective: To study the antibacteBursatellarial and a nleachiitiamyl asB.e pleachiiroperti, eKalingas of met hornataanol a nK.d aornatacetone, ( ) ( ) Dr. R. Sankar, Scientist, BCIA, Coastal eAplysiaxtracts of nudibranchs including Environmental Impact Assessment Methods:sp. D ivision, N ational C entre for Crude methanol and acetone extracts of sea slugs were tested for inhibition of fish Sustainable Coastal Management. bacterial pathogens' growth through disc diffusion method. The activity was measureαd based on E-mail: [email protected] the formation of inhibition zone around the disc impregnated with crude extracts. The -amylase Comments inhibitory effect was also measured calorimetrically. The chemical fingerprinting of the extract Results:was recorded with HPTLC and GC-MS. This is a good study, in which the The solvent extracts of all the three sea slugs showed antibacterial property. ThK.e ( 15 20 ) authors had evaluated the antibacterial mornataaximum zone of inhibition > Aplysia- m m was recorded for methanol and aαcetone extracts of 93 and alpha amylase inhibitory B. .leachii The m ethanol extract of K.s pornata. exhibited % inhibition against -amylase, following properties of solvent extracts of by (methanol) 70.6% and (methanol) 49.03% inhibition respectively. The nudibranchs. They made preliminary acetone extracts didn' t show any notabetc.le inhibition. The presence of free amino acids like lysine, analysis on the activities of the extracts aspartic acid, glutamic acid, arginine , terpenoids and pigents were confirmed through HPTLC GC MS and chemically fingerprinted the Conclusions:analysis. The p resence of siloxanes and propanoic acid were also revealB.ed leachiithrough K. -ornata. presence of various pharmacological Aplysia This study suggests that further scrutinisation of αthe , and targets for drug development. sp. will pave the way for development of antibacterial and -amylase inhibitory agent for Details on Page 191 therapeutic application. KEYWORDS α Antibacterial, -amylase inhibitory effect, Pigents and terpenoids 1. Introduction has picked up the rhythm in recent years with the growing recognition of their importance in human life[3]. Molluscs, New trends in drug discovery from natural sources which are widely distributed throughout the world, emphasize on investigation of the marine ecosystem to have many representatives in the marine and estuarine explore numerous complex and novel chemical entities. ecosystem[4]. Molluscs also feature prominently in a broad These entities are the source of new lead for treatment of range of traditional natural medicines though the active many diseases such as cancer, AIDS, inflammatory condition, ingredients in the taxa involved are typically unknown. arthritis, malaria and large variety of viral, bacterial, fungal Some marine gastropods and bivalves have been of great diseases[1,2]. Study of marine organisms for their bioactive interest to natural products chemists, yielding a diversity of potential, being an important part of marine ecosystem, chemical classes and several drug leads currently in clinical *Corresponding author: Dr. Arumugam Muthuvel.Faculty of Marine Sciences, CAS in Marine Article history: Biology, Annamalai University, Portonovo-608502, India. Received 25 Jul 2013 91 04144 243070 257 Tel: + - Ext: Received in revised form 3 Jul, 2nd revised form 8 Jul, 3rd revised form 13 Jul 2013 Fax: +91 04144-243555 Accepted 22 Aug 2013 E-mail: [email protected] ‘ Available online 28 Oct 2013 Founding Project: Supported by Ministry of Earth Sciences (MoES -G4/12319) under the Drugs from ’ the Sea programme. Giji Sadhasivam et al./Journal of Coastal Life Medicine 2013; 1(3): 186-192 187 [5] T 108 CFU L) C (100 trials . he rich diversity of marine organisms presumes µturbidity, approximately /m . ell suspension a great opportunity for the discovery of new bioactive L of target strain) was introduced into Muller Hinton agar M substances. any studies on bioactivity molecules with wide plates and spread finely on the plates using a glass spreadµer. range of activities like antitumor, antiviral, antimicrobial, The disc (6 mm, Wattmann No.1) was impregnated with 25 L anti-inflammatory were reported from Mollusca group. of sample (mg/mL) and standard amounts of the antibiotics as ( 10 ) The majority of molluscan natural products research is control amoxicillin, mg/mL . Then, the discs were p°laced focused within one of the major groups of gastropods, the on inoculated agar plates, which were incubated at 37 C for opisthobranchs (a subgroup of Heterobranchia), which 24 h. The degree of inhibition was determined by measuring are primarily comprised of soft-bodied marine molluscs. the diameter of zone of inhibition (in millimeters). M olluscs have an impressive array of defenses to protect 2.4. Alpha-amylase inhibition assay themselves from a broad array of predators from diverse taxa, including sea anemones, sea stars, crustaceans, fishes and humans[6]. Opisthobranchs are naked molluscs, apparently The amylase inhibitory activity was measured by following unprotected by the physical constrain of a shell. Sea Nickavar and Mosazadeh (2009)[8]. The starch solution hares, belonging to the order of opisthobranchia, subclass (0.5% w/v) was obtained by stirring and boiling 0.25 g of Gastropoda, are molluscs having many defense mechanisms soluble potato starch in 50 mL of deionised water for 15 T T 0 001 against their predators. he chemical defenses of these minα. he enzyme solution was prepared by mixing . g sessile organisms are built through the secretion of strongly of -amylase in 100 mL of 20 mmol/L sodium phosphate acidic substances, glandular secretions or inbuilt bioactive buffer (pH 6.9) containing 6.7 mmol/L sodium chloride. The compounds of their metabolism. As this marine mollusc extracts were dissolved in dimethylsulfoxide to give suitable represents a very interesting source of marine bioactive concentrations for the assay. The colour reagent was a molecules, the present study was carried out to prospect the solution containing 96 mmol/L 3,5-dinitrosalicylic acid (20 bioactive potential of three nudibranchs available in South mL), 5.31 mol/L sodium potassium tartrate in 2 mol/L sodium Indian waters. hydroxide (8 mL) and deionised water (12 mL). About 1 mL 1 of each the extracts and mL of the en°zyme solution were 25 C 30 A 2. Materials and methods mixed in a test tube and incubated at for min. bout 1 1 mL of this mixture was added to mL o°f the starch solution 25 C 3 T 1 2.1. Collection of samples and the tube was further incubated at for min. hen, mL of the c°olour reagent was added and the tube was placed 85 C A 15 Bursatella leachii B. leachii , into an water bath. fter min, the reaction mixture T ( ) 9 L Kalinga he n uornatadibra nK.ch ornataspecies, Aplysia[ was cooled and diluted with m distilled water and the ( ) sp.] were collected from absorbance value determined at 540 nm using a SHIMADZU the Mudasal Odai and Rameshwaram landing centre and UV-vis Spectrophotometer (Kyoto, Japan). Individual blanks transferred to laboratory in an ice box. The collected samples were prepared for correcting the background absorbance. In were washed thoroughly with tap and distilled water. The this case, the colour reagent solution was added before the samples were dissected °aseptically and chopped into small addition of starch solution and then the tube was placed into pieces and stored at -40 C until use. the water bath. Then, the method was followed as described C 2.2. Extraction above. ontrols were conducted in an identical manner, replacing extracts with 1 mL dimethylsulfoxide. Acarbose T solution was uαsed as positive control. he inhibition Tissue sample of 25 g of each was homogenized using percentage of -amylase was assessed by the following methanol (bioactive compounds extraction) and acetone formula. ° α 伊 - ( ) T 4 C 24 I % 100 (吤A C 吤A S ) 吤A C pigent extraction . he homogenate were kept in for -amylase = on-trol ample / ontrol- h extraction and then centrifuged at 3 000 r/min for 15 min. Where, 吤A Control=A Test A Blank, 吤A Sample=A Test A Pellets were re-suspended in respective solvents for re- Blank. extraction. The collected supernatants were subjected to ° 2.5. Characterisation rotary vacuum evaporator at 40 C. Further, the samples were lyophilised and stored until use. 2.5.1. High performance thin layer chromatography (HPTLC) 2.3. Antibacterial assay Qualitative chromatographic analyses were performed to identify the pigents,B. te rleachiipenoids K.an dornata amin o acidAplysias extrac ted The agar disc diffusion method was employed to screen the from nudibranchs , and sp. antibacterial activity of three nudibranchs extracts against Required (5 mg) amount of sample was dissolved in methanol B G 60 etei galht fish pathogenic bacterias as described by rumfitt and ethyl acetate s伊eparately and analysed on silica gel - (1990) [7] A TLC (10 10 ) (M D G ) . with slight modification . loop of bacterial plateµs cm cm erck, armstadt, ermany . 24 10 culture °inoculated into nutrient broth and incubated for About L samples were applied band wise onto the plates h at 37 C. The size was adjusted to 0.5 McFarland standard using the Linomat V automated TLC sampler III (Camag, Giji Sadhasivam et al./Journal of Coastal Life Medicine 2013; 1(3): 186-192 188 vietnamensis , Bacillus cereus, Halomonas Staphylococcus S ) TLC T ) witzerland on the stationary layer.
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