S244

Asian Pacific Journal of Tropical Biomedicine (2011)S244-S248

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Asian Pacific Journal of Tropical Biomedicine
journal homepage:www.elsevier.com/locate/apjtb

Document heading

Screening of antimicrobial potential of polysaccharide from cuttlebone and methanolic extract from body tissue of Sepia prashadi Winkworth, 1936
Pasiyappazham Ramasamy, Aruldhason Barwin Vino, Ramachandran Saravanan, Namasivayam Subhapradha, Vairamani Shanmugam, Annaian Shanmugam*
Centre of Advanced Study in Marine Biology Faculty of Marine Sciences, Annamalai University Parangipettai - 608 502, India

ARTICLE INFO
Article history: Received 15 August 2011 Received in revised form 8 September Accepted 1 October 2011 Available online 15 October 2011 Keywords: Antimicrobial activity Cephalopod Polysaccharide Cuttlebone

ABSTRACT Objective: To evaluate the antimicrobial activity of polysaccharide from cuttlebone and methanolic extract from body tissue of Sepia prashadi, against ten human pathogenic bacteria and five fungi. Methods: The activity of polysaccharide and methanolic extract was investigated against Vibrio cholerae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Vibrio alginolyticus, Staphylococcus aureus, Vibrio parahaemolyticus, Streptococcus sp., Streptococcus pneumoniae, Salmonella sp. and Escherichia coli, and five fungal strains such as Alternaria alternata, Candida tropicalis, Penicillium italicum, Fusarium equiseti and Candida albican using disc diffusion method and minimum inhibitory concentration (MIC) were also calculated. Results: Both polysaccharide and methanolic extract was active against gram positive than that of gram negative pathogenic bacteria but inactive against fungi. The MIC of both the extract ranging from 60 to 100 mg/mL. Conclusions: These results suggest that cephalopod polysaccharide and methanolic extract possess relatively good antibacterial activity.

2011

1. Introduction
The cephalopod which includes the Nautilus, cuttlefishes, squids and octopods, is the most advanced class of phylum: M ollusca adapted to a swimming existence. T hey are exclusively marine, diverse in form, size and nature[1] and occupy littoral and benthic to pelagic environments of all world oceans. Cephalopods are considerably important as a food resource as well as in scientific investigations. There is an ever continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action due to the alarming increase that has been witnessed in the incidence of both new and reemerging infectious diseases. A further big concern is the development of resistance to the antibiotics in current clinical use[2]. Although antibiotics are life saving drugs but nowadays, as a result of careless and promiscuous use of antibiotics, various pathogenic microbes are gaining resistances. A mong marine invertebrates, cephalopods belong to a

*Corresponding author: Annaian Shanmugam, Centre of Advanced Study in Marine Biology Faculty of Marine Sciences, Annamalai University Parangipettai - 608 502, India. Tel: +91-9443043597 E-mail: shanpappu48@gmail.com Foundation Project: Supported by Centre for Marine Living Resources and Ecology (CMLRE), Ministry of Earth Science (grant No. MoES/CMLRE/10A-4(6)/08).

molluscan group comprising 700 species in which bacterial associations have been known for a long time and can include the reproductive organs (accessory nidamental glands) of myopsids, sepiolids and sepiids and the light organ of sepiolids[3-5]. In recent years, human pathogenic microorganisms have developed resistance in response to the indiscriminate use of commercial antimicrobial drugs commonly employed in the treatment of infectious diseases. This situation, the undesirable side effect of certain antibiotics, and the emergence of previously uncommon infections, has forced scientists to look for new antimicrobial substitutions from various sources such as from plant origin and animal origin[6]. Marine invertebrates offer a source of potential antimicrobial drugs[7-9]. Discovered bioactive compounds in molluscs were identified essentially as peptide, depsipeptide, sterols, sesquiterpene, terpenes, polypropionate, nitrogenous compounds, macrolides, prostaglandins and fatty acid derivatives, sterols, miscellaneous compounds and alkaloids; they all presented specific types of activities[10-12]. Defer et al[13] reported the antibacterial and antiviral activities in three bivalve and two gastropod marine molluscs (Cerastoderma edule, Ruditapes philippinarum, Ostrea edulis, and Buccinum undatum). S tudies of antimicrobial activity provide valuable

Mueller Hinton agar (MHA. Gramnegative strains: Escherichia coli (E. I n our continuous search of antimicrobial agents from natural sources.4. Candida tropicalis. India. Annamalai University. Positive control disc containing 50 毺L of tetracycline (1 mg/mL) and as negative control. Inoculum preparation for bacteria Nutrient broth was prepared and sterilized in an autoclave at 15 lbs pressure for 15 min. alginolyticus). Sterile antimicrobial disc (Himedia) was impregnated with 50 毺L of polysaccharide and methanolic extract of the four concentrations tested. The sterilized Czapek dox agar was poured into sterile petridishes and incubated at 37曟 for 3 days.1. Czapek dox (Hi-media) broth was prepared and sterilized in an autoclave at 15 lbs pressure for 15min. The 24 hour-old bacterial broth cultures were inoculated in the petridishes by using a sterile cotton swab. T he P ublication of R oper et al. or extracts of whole body tissues have been tested for activity. The precipitate obtained after standing overnight was collected on Whatman No. Jothinayagam and Shanmugam et al were used in S245 Ten species of bacteria and five species of fungi were used as test organisms. The precipitate was dissolved in 10 mM EDTA solution and was dialyzed against deionized water. Penicillium italicum. coli). Result was calculated by measuring the zone of inhibition in millimeters. prashadi) were collected from Cuddalore landing centre ( L attitude 10曘42 ’ N . The stock solution of polysaccharide and methanolic extracts was prepared at a concentration of 100 mg/mL.8. Vibrio parahaemolyticus ( V. southeast coast of I ndia. hemolytic and surface activities. Klebsiella pneumoniae ( K. Then the methanolic extract was centrifuged to collect the supernatant and concentrated under vacuum in a rotary evaporator at low temperature. Due to the permanent resistance of the microorganisms to available drugs. 1 paper with hyflo supercel and washed with water. The bacterial strains contains Grampositive strains: Streptococcus sp. 2. prashadi).2. like haemolymph and egg masses. Annamalai Nagar. 2.3. Pseudomonas aeruginosa ( P. Fusarium equiseti and Candida albicans. the most common cause of enteric fever. These impregnated discs were allowed to dry at laminar air flow chamber for 3 hours. With an estimated 16-33 million cases resulting in 500 000 to 600 000 deaths annually in endemic areas. 1 filter paper with hyflo super-cel. phylogenetic analysis and screening of marine mollusc-associated bacteria for antimicrobial. body tissues were removed. Inoculum preparation for fungi identification[16-18]. this study was designed to assess the antimicrobial activity of the polysaccharides extracted from cuttlebone and methanolic extract from body tissue of Sepia prashadi (S. The air dried cuttlebones were pulverized and washed with acetone. 50 毺L of 10 mM EDTA and methanol were used. 2.6. Preparation of polysaccharide extract from cuttlebone The polysaccharide extact was prepared using the method described by Okutani et al[19]. 2. In most of the publications concerning antimicrobial activity in molluscs. which also includes paratyphoid fever caused by Salmonella paratyphi. Vibrio alginolyticus (V. RQ-127 A) and extracted with MeOH at room temperature fore 24-48 h. and were placed at the respective bacterial and fungal plates and incubated at 37曟 for 24 h for bacteria and 72 hours for fungi. Salmonella enterica serovar Typhi. Staphylococcus aureus (S. cut into small pieces and homogenized (REMI.5. The dialysate was freeze-dried which was then used in the present investigation. All the bacterial and fungal strains were clinical isolates. obtained from the Raja Muthaiah Medical College Hospital. All the ten bacterial strains were individually inoculated in the sterilized nutrient broth and incubated at 37 曟 for 24 h. The diameter (mm) of the growth inhibition halos produced by the polysaccharide and methanolic extract of cephalopods was examined. 2. All the tests were performed in triplicate. Himedia) was prepared. sterilized in an autoclave at 15 lbs pressure for 15 minutes and poured into sterile petridishes and incubated at 37 曟 for 24 h.. Determination of the minimum inhibitory concentration 2. Vibrio cholerae (V. The powder was extracted with hot 10 mM EDTA solution and filtered using Whatman No.. continuous search for new antimicrobials is a scientific challenge.Pasiyappazham Ramasamy et al. either single body component alone. The fungal strains contains Alternaria alternata. Romanenko et al[14] investigated the isolation. The 72 hour -old fungal broth cultures were inoculated in the petridishes using a sterile cotton swab. Microbial cultures The polysaccharide and methanolic extracts were selected for the determination of MIC following the method of . aeruginosa ) . Antibacterial and antifungal activity was determined following the method of El-Masry[21]. Preparation of methanolic extracts from the body tissues The methanolic extracts of the body tissues were prepared by following the method of Ely et al[20]. prashadi were brought to laboratory. cholerae). Five fungal strains were inoculated in the broth and incubated at 37曟 for 72 h. Typhoid fever is a more classical systemic infection caused by the typhoid bacillus. 2.7. S. Streptococcus pneumoniae (S./Asian Pacific Journal of Tropical Biomedicine (2011)S244-S248 information for new antibiotic discoveries and give new insights into extraction of bioactive compounds from aquacultured molluscs. Disc diffusion method 2. Then saturated barium hydroxide solution was added to the filtrate. Materials and methods 2. the WHO identifies typhoid as a serious public health problem[15]. aureus). Sampling and identification The specimen (S. pneumoniae) and Salmonella sp. parahaemolyticus ) . pneumoniae). L ongitude 79曘46 ’ E ) .

82 7暲0.53 9暲0.82 11暲1.58 9暲0. parahaemolyticus Streptococcus sp.82) mm was recorded against Streptococcus sp. pneumoniae V.1. The lowest activity with (7暲 mm inhibition zone was observed against Salmonella sp.58 9暲0.58 8暲0. in methanolic extract. V.53 11暲0. coli 100 + * + * + + ++ +++ ++ + + ++ 60 +++ ++ ++ ++ ++ + +++ 40 +++ +++ +++ 20 100 * + + + 80 + Methanolic extracts (mg/mL) +++ ++ 60 ++ ++ ++ ++ * * * +++ +++ +++ ++ ++ * +++ +++ +++ + +++ +++ +++ +++ +++ +++ ++ * * + ++ +++ + +++ +++ 40 * + + +++ ++ +++ ++ +++ +++ 20 +++ +++ +++ +++ +++ +++ +++ ++ + +++ ++ ++ +++ +++ +++ +++ +++ +++ +++ +++ * ++ . prashadi showed antimicrobial activity against all pathogenic bacteria.No growth. pneumoniae in polysaccharide extract and (7暲 0. Bacterial strains * MIC concentration. pneumoniae Salmonella sp. aureus V.58 10暲0.25 22暲1. maximum activity of (8暲0.58 11暲0.58) mm against S. parahaemolyticus and Streptococcus sp.2. aeruginosa K. no activity was recorded against all the fungal strains studied. aureus.82 7暲0. the highest inhibition zone of (13 暲 1 . S. cholerae.58 9暲1. 100. aureus in methanolic extract. V..82) mm against K. parahaemolyticus 7暲0. + Cloudy solution (slight growth).82 8暲0. alginolyticus.58 - Table 2 MIC of polysaccharide and methanolic extracts of S.05 were considered for describing the significant levels. Bacterial strains 25% conc 7暲 0. cholerae.58 9暲0. lowest in 25% concentration and there was no activity in negative control (Table 1). 60 and 80 mg/mL respectively. In 100% concentration.25 7暲0. but at the same time they didn’t show any activity against fungi.82 12暲1.05 (DMRT). 100.25 7暲0. pnemoniae in methanolic extract. 80. aureus. alginolyticus 8暲0. V.58) mm against V. In 75% concentration.25 8暲0.58 10暲0. aureus V.25 9暲0. aeruginosa. 80. In 25% concentration. aureus. aureus in methanolic extract.82 12暲1. pneumoniae in methanolic extract.82 10暲0. aureus in polysaccharide extract and (7 暲0. ++ Turbid solution (strong growth).58 V. The lowest activity of (7暲0. MIC of the active extract against the test organisms T he MIC results are given in T able 2 . S. in polysaccharide extract and (7暲0. were reported as 80.82) mm was observed against S.58 29暲1.82 7暲0.58 Streptococcus sp.58 27暲1.53 12暲1. The activity was highest in 100% concentration.58 10暲1.82 11暲0.82 10暲0.58) mm was recorded against S. parahaemolyticus and S.25) mm inhibition zone was recorded against S. pneumoniae and E.58 8暲0. The lowest inhibition zone of (8暲0.82 18暲0. Whereas for methanolic extract. 3. coli - mm for methanolic extract. MIC values of polysaccharide against bacterial strains such as V. But at the same time. coli was recorded as 100. pneumoniae. E.25 13暲1.53 7暲0. aeruginosa K. S.53 9暲0.82 10暲1..58 10暲0. aureus and (10暲1. +++ Highly turbid solution (dense growth).53) Table 1 Antibacterial activity of S.00 11暲0. parahaemolyticus in polysaccharide extract./Asian Pacific Journal of Tropical Biomedicine (2011)S244-S248 Data on the inhibitory effects of polysaccharide and methanolic extracts of cephalopods was analyzed by oneway analysis of variance (ANOVA) using SPSS-16 version software followed by Duncun’s multiple range test (DMRT) and Mean 暲 SEM.58 9暲0. E.58 10暲1.82) mm against S. the MIC for V.58) mm against Salmonella sp.58 8暲0. . prashadi against tested microorganisms. cholerae P. V.82 28暲1.58 10暲0. Polysaccharide extracts (mg/mL) 80 ++ Inhibition zone in methanolic extract form Positive control Negative control from cuttlebone (mm) body tissues (mm) (Tetracycline) (Methanol & EDTA) (mm) (mm) 50 % conc 75% conc 100% conc 25% conc 50 conc 75% conc 100 % conc 9暲0. Results The polysaccharide and methanolic extracts of S. and (9暲0.00 12暲1.82 24暲1. S. was noticed for methanolic extract. Statistical analysis Rajendran and Ramakrishnan[22].58 S. the maximum inhibition zone of (11 暲0.53 7暲0. S246 Pasiyappazham Ramasamy et al.58 9暲0. 53 ) mm was noticed against V. K.82 11暲1. prashadi.25 26暲1. 0. 100 and 100 mg/mL respectively. V. parahaemolyticus. Inhibition zone in polysaccharide extract conc: Conncentration. pneumoniae V. P values at <0.9.58 8暲0. alginolyticus in polysaccharide extract and same level of inhibition against P. polysaccharide extract showed highest activity of (11暲1. and (12暲1.58) 3. V.25 24暲1.25 7暲0. pneumoniae Salmonella sp.58 23暲1. 80. In 50% concentration.58 8暲0.82 7暲0. alginolyticus S. in polysaccharide extract.58 9暲0. cholerae P.58 10暲0.00) mm against S.53 9暲0. S. alginolyticus and Streptococcus sp. * The statistical significance: P values <0.53 7暲0.

vitellas[27] . aureus and 7 mm against Salmonella typhi (S. In the present study the activity was also dose dependent. cholerae. there exists antibacterial against different bacterial strains. pneumoniae. V. the polysaccharide extract from the cuttlebone reported (13暲 1.. coli and K. aeruginosa. Discussion In recent years. Patterson and Murugan reported the broad spectrum of antibacterial activity for aqueous extract of ink of the cephalopods Loligo duvaucelii ( L. However the majority of marine organisms are yet to be screened for discovering useful antibiotics. The polysaccharide extract from the gladius of D. polysaccharides (EDTA) extract from S. prashadi.e. fumigatus and Rhizopus sp. The overall objective of the current study was to compare the ability of antibacterial and antifungal activity of methanolic extract of the body tissue and polysaccharide extract from the cuttlebone of S. Membrane disruption could be suggested as one of the likely mechanisms of action of friedelin[35]. 9 mm inhibition zone against S. 4 mm against S. S. mussel (Mytilus edulis and Geukensia demissa). duvaucelii ) and Sepia pharaonis against nine human pathogens[28]. But at the same time no antifungal activity was recorded in both polysaccharide and methanolic extracts studied. But at the same time both the species showed no activity against Candida sp. brevimana. E. typhii) (excluding the disc size of 5 mm dia). At the same time. fungi and yeasts[23]. Further investigation on the purification and chemical elucidation of the active principle present in both the extracts shall pave the way for the development of either the base or a new drug itself in future. The activity was recorded in almost all the concentrations except in negative control. a low dose of a ‘bactericidal’ antibacterial agent may only inhibit bacterial growth. Conflict of interest statement We declare that we have no conflict of interest. duvauceli gladius showed only low activity i. This highest activity of polysaccharides may be due to the destruction of cell wall of V.53) mm of inhibition zone against V. typhi and E. which indicated that cell wall and membrane of S. C. in laboratory studies and natural environment while may be due to their varying concentration present in the extracts used in both the places[33. prashadi cuttlebone exhibited better activity. pnemoniae. sibogae recorded potent antibacterial activity against all the bacterial strains mentioned above and at the same time the polysaccharide of the L. This result was consistent with that of oleoyl-chitosan. C. cholerae. In the present investigation. brevimana) against almost all the 9 pathogenic bacterial strains tested viz. aeruginosa and Shigella sp. Further the dose as well as the concentration of the active principle in the extract shows either ‘bactericidal’ or ‘bacteriostatic’ action against the bacteria i. only. they indicated the high frequency of detectable antimicrobial activity in marine molluscs. S247 . aureus. fumigatus). Bacillus subtilis. parahaemolyticus. prashadi. tigris and C. flavus) and Rhizopus sp. Several molecules extracted from marine invertebrates. Antibacterial activity has previously been described in a wide range of molluscan species such as oyster (Crassostrea virginica). Whereas the EDTA extract of L. T he polysaccharide extract from the gladius of L. sibogae) gladius reported 10 mm inhibition zone against E. at all the concentrations tested. K. S. great attention has been paid to the bioactivity of natural products because of their potential pharmacological utilization./Asian Pacific Journal of Tropical Biomedicine (2011)S244-S248 4. sibogae reported activity against A. parahaemolyticus and the methanolic extract from the body tissue showed (12暲1. The cuttlebone extract of S. There are only a few studies carried out hitherto on the antibacterial activity of the internal bone of cephalopods. muricid mollusc (Dicathais orbita) and sea hare (Dolabella auricularia ) [24-26]. arabica. typhi. duvauceli showed activity against the fungi such as Aspergillus fumigatus (A. aculeata than S. prashadi which explains and supports the presence of active principle in both the methanolic and polysaccharide extracts obtained from the body tissue and cuttlebone of S. On comparison the maximum zone of inhibition was obtained for the methanolic extract than the polysaccharide extract from S. while a high dose of a ‘bacteriostatic’ antibacterial agent will be bactericidal[36]. Premanand and Patterson also reported no antifungal activity in the methanolic extracts from Didemnum psammathodes[27]. aculeata and S. the gladius extract of both the species showed no activity against V. Candida sp. for the EDTA extract (polysaccharides ) of Doryteuthis sibogae (D. In the present study a wide spectral antibacterial activity has been recorded in almost all the concentrations. C. Although different species and experimental procedures were used in the different studies.e. whereas gladius extract of D. The known antimicrobial mechanisms associated to each group of chemical to which the isolated compounds belong may explain the antimicrobial potency of the crude extracts and compounds from Vismia laurentii. parahaemolyticus and S. Water-soluble intracellular protein of S. duvaucelii gladius extract recorded only low activity. Most homeopathic medicines are either of plant or animal origin. These results enforce the idea that cephalopods are a source to be considered in the discovery of new substances for drug development. aculeata) and Sepia brevimana (S. Aspergillus flavus (A.. possess broad spectrum antimicrobial activities.25) mm of inhibition zone against S. which could destroy cell wall of S. V. On comparison the activity was higher in the cuttlebone extract of S. onyx. P. the zone of inhibition was relatively higher than that of the previous above study. flavus. showed the maximum antifungal activity in 100% concentration and the activity was found to be in an increasing order from the lower to higher concentration. and Rhizopus sp. aureus treated with the polysaccharide increased rapidly. At the same time. fumigatus. coli.34]. coli (excluding the disc size of 5mm dia). affecting the growth of bacteria. aureus. aureus. But in the present study at 100% concentration. 5 mm against P.. Shanmugam et al[30] observed that the antibacterial activity was predominant among cuttlebone extracts (using EDTA) of the cuttlefishes such as Sepia aculeata (S. including bivalves. errones. aureus might be disrupted by the polysaccharide [32]. H owever there exists a difference in the activity shown by the compound(s) present in the extracts.Pasiyappazham Ramasamy et al. Barwin Vino[29]. P rem A nand and P atterson E dward reported moderate antibacterial and antifungal activity from the extracts of five species of Cypraea namely C. aureus and lead to intracellular components being released[31]. brevimana against four fungal stains such as A. A. The results of the present study clearly showed that the 75 % and 100 % concentrations exhibited appreciable antibacterial activity against human pathogens (Tables 1 & 2). W hen compared to the above mentioned studies.

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