Journal of Coastal Life Medicine 2013; 1(3): 186-192 186

Journal of Coastal Life Medicine

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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 an leachiitiamyl 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 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 ), 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 , 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. he plates were Bacillus sp., CAMAG HPTLC T developed in a vapour equilibratedn twin sp., sp. he maximum area of zone of inhibition ( 10 ) 3 1 trough chamber about min using -henxane:acetone : wasK. o bornataserved for the Staphylococcuscrude acetone andBacillus methanol extracB.ts ( ) v/v as a mobile phase for pigents and -butanol:acetic vietnamensisof against sp, sp and 1 1 1 1 ( ) acid:ethyl acetate:water : : : v/v for bioactive compounds. whereasB. th eleachii minim um activity was obsB.er vflexused for A 5 ( ) fter development, the plates were air dried for min, and Halomonasthe crude extract of Aplysia methanol against , sprayed with ninhydrin for visualisation of amino acids and sp. and sp. Halomonasexhibited limited activity with p-anisaldehyde for terpenoids (brown ring confirmation (acetone and methanol) against sp. The results test was also done). Pigents were visualised using UV lamp were tabulated (Table 2). D T CAMAG in ensitometer. he plates were then scanned using 3.3. Alpha-amylase inhibition assay TLC scanner 3 winCATS software (version 4X) for the presence Rf of pigents, terpenoids and amino acids using the values α and images. The -amylase inhibitory activity was measured by using reducing sugar method. The crude methanol extract of 2.5.2. Gas chromatography-mass spectrometer (GC-MS) Aplysia 93% analysis B. leachiisp . exhibited the maximαum activity of , whereas (methanol) arrested -amylase activity by 70.6%. The sample was injected in GC column (Accu-TOF GCV- The methanol extract of Kalinga inhibited moderately around JMS T100GCV JEOL ) 49 03% - , Asia, J°apan . The column temperature . respectively. Thus methanol extracts from 80 280 C 20 was maintained at - , the split ratio was and the under investigation showed alph’a amylase inhibition activity helium flow rate was maintained at 1 mL/min. Then the whereas acetone extracts didn t show any inhibitory effect possible groups were identified for the sample peaks using (Figure 1). MS library. 100 90 80

3. Results n o i

t 70 i b i 60 h

3.1. Extraction of bioactive compounds n I

50 f o 40 % 30 The wet weight and net yield of the methanol and acetone 20 extracts of nudibranchs were tabulated (Table 1). 10 0 Table 1 Methanol Acetone Solvent extract Aplysia B. leachii K. ornata The yield of all the extracts from the fresh were tabulated. spp. Organism Source Solvent Wet Weight (g) Net yield (g) Figure 1. α B. leachii The -Amylase inhibitory activity of methanol and acetone extracts Body Acetone 7.47 0.128 1 of nudibranchs. Body Methanol 63.25 0.664 0 3.4. HPTLC characterisation of extracts K. ornata Body Acetone 36.04 0.325 7 Body Methanol 5.00 0.098 9 3.4.1. Pigent analysis Aplysia B A 108 07 3 094 3 sp. ody cetone . . The presence of pigents was identified at 280 nm. Acetone Body Methanol 5.00 0.101 2 280 3.2. Antibacterial assay extracts showed significant intense peak at nm while methanol extracts showed slight absorbance when scanned using CAMAG scanner 3. Nevertheless, the magnitude of T T he antibacterial activity was tested usiBacillusng agar d firmus,iffusion pigents distribution varied depending upon the species. he ( F 2 mBacillusethod a gflexusainst sB.ev eflexusn fish ,p Bacillusathogens vietnamensis B. absorbance spectrum of samples is shown in igure . ( ) ( The peaks from a to e show the presence of pigents in Table 2 Antibacterial activity of nudibranch extracts against bacterial fish pathogens. Pathogen name Bacillus firmus B. flexus B. vietnamensis Bacillus cereus Halomonas Staphylococcus Bacillus Samples Tested sp. sp. sp. Zone of inhibition in mm B. leachii Methanol 16 12 14 12 12 17 15 B. leachii Acetone 12 10 15 11 09 14 13 K. ornata Methanol 17 19 16 18 14 23 20 K. ornata Acetone 19 17 20 17 16 21 18 Aplysia spp. Methanol 14 12 13 09 10 16 13 Aplysia spp. Acetone 12 11 13 11 10 12 12 Control 24 15 25 20 19 16 17 Giji Sadhasivam et al./Journal of Coastal Life Medicine 2013; 1(3): 186-192 189 R All tracks @ 280 nm 600.0 f (F 4) [AU] corresponding values of standards and samples igure 400.0 whereas the other components remain unknown which may 300.0 200.0 be due to the presence of impurities and other non standard 100.0 0.0 amino acids. The list of amino acids present in the sample 100 (T 3) wTableere t a3bulated able . a [mm] Shows the free amino acid analyses in the nudibranchs extract. b 80.0 R Sample f Possibile amino acids in sample by comparing R 1 2 3 70.0 peaks with standard f value c 0.22 Arginine, histidine, lysine * * * 60.0 0.29 Cysteine * * - d 50.0 Glycine, aspartic acid, glutamine, proline, 0.45 * * * aspargine, serine, hydroxyproline 40.0 e 0.62 Glutamic acid, threonine - * * 30.0 0.75 Valine, methionine * * * 0.92 Tyrosine, tryptophan, phenyl-alanine, leucine * * * 600.0 20.0 [AU] f 400.0 3.4.4. GC-MS 300.0 10.0 200.0 Interpretation on mass spectrum of GC-MS was conducted 100 0 . 0.0 0.0 R using the database of National Institute of Standard and 0 20 0 00 0 20 0 40 0 60 f 1 00 - . . . . . [ ] . Technology (2005) having more than 62 000 patterns. The Figure 2. Spectrum shown by the pigents at 280 nm when scanned with Camag spectrum of the separated components was compared 3 with the spectrum of National Institute of Standard and scanner . B. leachii, K. ornata, Technology library database. The identity of the spectra Aplysiamethan ol and acetone extracts of spp. should be above 95% for the identification of components. The methanol extracts were analysed with GC-MS to identify 3.4.2. Terpenoids analysis the possible bioactive molecules. Extracts showed so The presence of terpenoids was confirmed at two levels. The many junk peaks along with our compounds of interest, preliminary qualitative analysis of terpenoids was carried out since it was a crude sample. Siloxane was the major group using brown ring formation test (Salkowski test) and further commonly found in all the methanol extracts next to that substantiated by HPTLC analysis. Terpenoids present in the propanoic acid which was commonly present in all the sample showed purple spots when sprayed with anisaldehyde samples. Besides this, some of the major peaks contributed Rf 2 ( ) 2 4 (1 reagent. Terpenoids ( : 0.94) were detected only in the to the presence of, - methB.yl leachiithio , phenol, , -Aplysiabis , methanol extracts of nudibranch (Figure 3). The difference in 1-dimethyl ethyl) in sample . Whereas in their intensity supports their diverse presence in the extract. sp. peaks responsible for the presence of (1, 3) thiazinane- 4-carboxylic acid, 3-methyl and 2, 2, 5-trimethyl 3.4.3. Free amino acids analysis - K. ornata piperidin-3-ol were recorded. contains glycine, The presence of free amino acids in the samples was N-(methoxyoxoacetyl)-, methyl ester, (1, 3) thiazinane-4- confirmed through HPTLC in comparison with standard amino carboxylic acid, 3-methyl methyl tetradecanoate. The GC- acids. Among the 21 amino acids, few of them were detected MS of the methanol extracts of nudibranchs were shown in in the nudibranchs extract which was confirmed by their Figure 5. Track Track 2 Track 4 300 400 AU 1 AU 2 500 3 AU 350 450 250 400 300

200 350 250 300

150 1 200 250 1

150 200 100 150 100 1 100 50 50 50

0 R 0 R 0 R 0.11 0.09 0.29 0.49 0.69 0.89 f 0.11 0.09 0.29 0.49 0.69 0.89 f 0.11 0.09 0.29 0.49 0.69 0.89 f Figure 3. B. leachii K. ornata Aplysia sp Shows the peak responsible for terpenoids when scanned at 463 nm. 1. methanol, 2. methanol, 3. . methanol. (Photos on the left side depicts brown ring formation responsible for terpenoids in the sample). Giji Sadhasivam et al./Journal of Coastal Life Medicine 2013; 1(3): 186-192 190 Track 2 .ID: Track 3 .ID: 400 400 200 AU 1 AU 2 AU 3 350 350

300 12 13 300 150 11 9 10 250 10 250 8

200 100 200 13 12 1011 13 7 3 9 150 9 2 141516 17 8 150 6 7 5 6 7 8 18 19 6 12 1 4 5 100 5 11 50 100 3 4 20 4 14 18 3 16 2 14 2 17 19 1 15 50 1 15 50

0 0 0 -0.11 0.09 0.29 0.49 0.69 0.89 Rf -0.11 0.09 0.29 0.49 0.69 0.89 Rf -0.11 0.09 0.29 0.49 0.69 0.89 Rf Figure 4. B. leachii K. ornata Aplysia Sp HPTLC analysis of free amino acids of Nudi extracts. 1) methanol, 2) methanol, 3) s methanol

Experiment Date/Time:4/19/2012 11:34:24 AM 3 伊10 Intensity(442988) 21 Australian dorid nudibranchs extracts and reported that 18 3 400 . A antimicrobial activity was present 80% of the extracts[13]. 300 Aplysia californica 9 5 Also the protein isolated from the ink of 200 3.1 6.5 . 6.5 12.4 inhibited the growth of gram positive and gram negative 100 9 6 . [14] 0 2 4 6 8 10 12 14 16 18 20 22 bacteria including marine bacteria, yeast and fungi . Time[min] Experiment Date/Time:4/19/2012 11:34:24 AM 3 Similarly in our study, all the nudi samples showed 伊10 Intensity(555443) 23.0 400 considerable inhibition against all the fish pathogens B K. ornata 21.1 tested. In which, sample showed significant

200 20.8 inhibition potency. Thus, the current result proved that our 11.6 4 2 14.4 3.1 . antibacterial agents possess a broad spectrum of activity in 0 0 2 4 6 8 10 12 14 16 18 20 22 nature. Experiment Date/Time:4/19/2012 11:34:24 AM Time[min] 3 伊10 Intensity(442988) 18.3 Obesity, and resultant health hazards which include 400 C 300 diabetes, cardiovascular disease and metabolic syndrome, 9.5 200 3.1 6.5 are worldwide medical problems. Control of diet and 12.4 100 6.5 9.6 exercise are cornerstones of the management of excess 0 [15] 0 2 4 6 8 10 12 14 16 18 20 22 weight . Amylases are a class of enzymes that hydrolyze Time[min] Figure 5. B. leachii starch to yield low molecular weight dextrins and sugars and A) GC-MS spectrum of methanol extract; B) GC-MS spectrum K. ornata Aplysia they play important role in the digestion of carbohydrates. of methanol extract; C) GC-MS spectrum of sp. methanol extract. α α Inhibition of -amylase along with -glucosidase can significantly reduce the post-prandial increase of blood 4. Discussion glucose and can be an important strategy in the control of blood glucose level in the type-2 diabetic patients. Hence, I pancreatic a amylase and gastric glucoamylases are the n the last three decades, the study of marine chemical [16] ecology has experienced great progress due to the new major therapeutic targets for the type-2 diabetes mellitus . technological advances for collecting and studying marine About 246 million people suffer from type-II DM worldwide, samples[9,10]. The largely unexplored marine world that and its incidence and serious complications continue to grow rapidly. Although there are several classes of antidiabetic presumably harbors the mos‘t biodiver’sity may be the vastest resource to discover novel validated structures with novel drugs, achieving and maintaining long-term glycemia modes of action that cover biologically relevant chemical control is often challenging. In addition, many current [17] I space[11]. Thus, knowing the importance of sea hare in the αagents have treatment-limiting side effects . nhibition of development of new bioactive peptides, we have attempted a -amylase, enzyme that plays a role in digestion of starch ( ) and glycogen, is considered a strategy for the treatment study to reveal the potency of acetone pigents and methanB.ol (bioactive molecule) extracts of three different sea hares of disorders in carbohydrate uptake such as diabetes and leachii K. ornata Aplysia [18] , and sp. obesity as well as dental caries and periodontal diseases . The present study proved that both methanol and acetone Besides this, due to the gastro intestinal side effects extracts were potent antibacterial agents against fish exhibited by oral anti-hyperglycaemic agents, searching pathogens investigated. The maximum area of inhibition for new amylase inhibitors became essential in treatment H zone was observed for the crude acetone and methanol of diabetes. ence, in the pαresent study, the potency of K. ornata Staphylococcus Bacillus nudi extracts were tested for -amylase inhibition activity extracts B.of vietnamensis agaiStaphylococcusnst sp., Aplysia sp. and , sp. respectively. where methanol extract oD.f auriculariasp. showeK.d ornatastrong ( ) F inhibition 93% , whereas , and urthermoDolabellare, the an tauriculariaibacterial a cD.tiv iauriculariaty of purple fluid of ( ) showed average inhibition. Similarly, Abirami and her sea hare et al against ’ A [12] E colleagues have reported alpha amylase s inhibitory effect human pathogens was reported by birami . arlier, D. auricularia[12] et al. Gunthorpe and Cameron screened bioactive properties of in the purple fluid of . Further, Ravi Giji Sadhasivam et al./Journal of Coastal Life Medicine 2013; 1(3): 186-192 191

72% GC-MS GC-MS observed alpha amylase inhibitory aHemifususctivity of pugilinus in acetone extracts were analysed with . results revealed Naticaextract sdidyma of two marine gastropods, ’ and the presence of siloxane, a chemical compound which has , whereas, here acetone etex al.tracts didn t show various applications found in products such as cosmetics, notable results[19]. Furthermore, Tiwari established the deodorant, water repelling windshield coating, lubricants, B anti hypScapharcaerglycemic activity of the crude extract of bivalve molded lenses, food additives, and some soaps. esides this, mollusc inaequivalvisin rat models[20]. peak responsible for the occurrence of propanoic acid, a Marine animals especially those from tropical waters are naturally occurring carboxylic acid was recorded. It is used often brilliantly coloured, and this is widespread in both to make pesticides and preservatives in pharmaceutical sessile and non-sessile invertebrates where distribution industries, as it inhibits the growth of mold and some and function of pigents seem to vary between invertebrate bacteria. This substantiates that propanoic acid may be [21] T B.gro leachiiups . K.h eornata crude acetAplysiaone an d methanol extracts of one of the contributing factors for the antibacterial activity , and sp. were tested for the exhibited by the nudibranch extracts. presence of pigents, terpenoids and amino acid analysis Nudibranchs being a slow moving secrete HPTLC through K. b yornata comparing with those of standards. bioactive molecules either for defence or prey capture. A T ccordinAplysiagly, showed orange pigented spots hus, it represents as a source of bioactive compounds whereas sp. showed dark green spots through out the to be explored. Hence, this present study would gain us C K.bo dornatay. onsequeAplysiantly, th e intensity of the peaks recorded for knowledge about theB. b ileachii,o-pote nK.cy ornata of cru de aAplysiacetone an.d D. auricularia and sp.were high when compared to that methanol extracts of and sp , of pigents in the crude extracts of the samples which may pave a way for the isolation of specific protein identified based on their spectral absorbance pattern at or novel compounds and/or development of new therapeutic 280 nm. The presence of amino acids in the crude extracts strategies complementary to conventional therapy and also were confirmed through comparison with standard amino imply that further investigation on the bioactive potential acids. Commonly, glycine, aspartic acid, glutamine proline, of this sea slugs will provide more bioactive molecules of aspargenine, serine, hydroxyproline, tyrosine, tryptophan, biomedical impact. phenylalanine, leucine were present in all the methanol extracts of samples. Some amino acids were exceptionally Conflict of interest statement present in specific samples. Apart from the 22 standard amino acid, some bands were observed which may be due to the presence of non standard amino acids. Some amino We declare that we have no conflict of interest. acids have identical migration point when used in particular [22] solvents which are supported by Carol and Prescott . As Acknowledgements stated above, some amino acids in the extracts have same migration point and could not be identified distinctly. Terpenoids represent a promising and expanding The authors are indebted with Ministry of Ear‘th Sciences ( 4 12319) source for biologically active natural compounds whose MoES -’G / for financial support under the Drugs from potential for research and development of new substances the Sea programme. We also thank Annamalai University with pharmacologic activity[18]. There have been many for providing us necessary facilities. applications of terpenes in human societies. Pharmaceutical and food industries have exploited them for their potentials Comments and effectiveness as medicines and flavour enhancers[23]. T he presence of terpenoids in the crude extracts wB.as Background 463 T dleachiietectedK. a ornatat nm. Aplysiahe cru de methanol extracRts of , and sp. showed peak at f value It is accredited that marine invertebrates produce 0.94 which shows the presence of terpenoids. Terpenes bioactive natural products that may be useful for developing H represent the major group of secondaetry al.metabolites isolated new drugs. owever, mining the marine environment will from opisthobranchs. Earlier Souza reported the role clear the way for chemical and biological novelties as of terpenoids in antimicrobial activity against cariogenic well. Hence, bioprospecting in marine has potential and bacteria[24]. This greatly supports our results on the role promising outcome. M of terpenoids in biological activities tested. ost of these Research frontiers compounds show both interesting biological properties and unique chemical structures. In particular, diterpenes The reported research work portrayed that the solvent include some of the most interesting examples of bioactive extracts exhibited significant inhibition against fish molecules; hence, the occurrence of terpenoids was tested in pathogens and alpha amylase enzyme. Further, the extracts the crude methanol extracts where all the samples showed were well characterized and correlated with the bioactivity the presence of terpenoids at notable level. Hence, through of the extract investigated. HPLTC , the presence of pigents, terpenoids, amino acids were Related reports confirmed in the crude extracts of nudibranchs. Besides this, to identify the other bioactive molecules, the Earlier crozier identified the presence of pigents in Giji Sadhasivam et al./Journal of Coastal Life Medicine 2013; 1(3): 186-192 192 Chronodoris zebra Heilprin, the largest of the nudibralichs [5] B enkendorff K. Molluscan biological and chemical diversity: B R (2002) secondary metabolites and medicinal resources produced by found in ermuda. ajaganapB.at hleachiii Aplysiaidentifi edactylomelad the anti- Biol Rev Camb Philos Soc 85 HIV activity of purple ink of . marine molluscs. 2010; (4): 757-775. ink has shown antimicrobial activities that inhibited the [6] N usnbaum M, Derby CD. Effects of sea hare ink secretion and its escapin-generated components on a variety of predatory fishes. growth of bacteria, as well as being toxic to many larger Biol Bull 218 - organisms such as brine shrimp (Melo VMM, 1998). 2010; (3): 282 292. [7] B rumfitt W, Hamilton-Miller JM, Franklin I. Antibiotic activity of Innovations and breakthroughs Microbios 62 natural products: 1. Propolis. 1990; (250): 19-22. T [8] N ickavar B, Mosazadeh G. Influence of three morus species he marine bioprospecting is the promising field in α Iran J Pharm Res 8 discovery of natural products. The opisthobranchs of the extracts on -amylase activity. 2009; (2): 115- tropical and temperate Pacific are remarkably diverse, 119. [9] P aul VJ, Ritson-Williams R, Sharp K. Marine chemical ecology in which were reviewed in a series of regional monographs on Nat Prod Rep 28 Hawaii (Gosliner 1980), South Africa (Gosliner and Griffiths benthic environments. 2011; (2): 345-387. 1981) I O S P [10] B lunt JW, Copp BR, Keyzers RA, Munro MH, Prinsep MR. Marine , and the ndian cean and the outh-west acific Nat Prod Rep 30 (Miller 1971, 1974, 2001, Rudman 1980, Schrodl 2003). Hence natural products. 2013; (2): 237-323. I [11] M ontaser R, Luesch H. Marine natural products: a new wave of the nudibranch collected from tropical waters of ndian Future Med Chem 3 coast will have different metabolites depending on their drugs? 2011; (12): 1475-1489. predation and feeding mechanism. Further, identification of [12] A birami P, Arumugam M, Ajithkumar TT, Balasubramaniam T. 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