Antimicrobial activity of methanolic extract and fractionated polysaccharide from Loligo duvauceli Orbingy 1848 and Doryteuthis sibogae Adam 1954 on human pathogenic microorganisms

In the present study, in vitro antimicrobial activity of crude methanolic extract of squid body tissue and fractionated gladius polysaccharide of two squid (Loligo duvauceli and Doryteuthis sibogae) from Cuddalore and Mudasalodai (Southeast Coast of India) landing centre was evaluated. The antimicrobial activity was screened against nine species of clinically isolated human pathogenic bacteria namely Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Vibrio cholerae, V. parahaemolyticus, Staphylococcus aerues, Pseudomonas aeruginosa, Salmonella typhii and Shigella sp. and four fungal strains such as Candida sp., Rhizopus sp., Aspergillus flavus and A. fumigates. Different concentrations such as 25, 50, 75 and 100% were prepared and tested against the microbial strains for their inhibitory activities, using the disc diffusion method. The minimum inhibitory concentration (MIC) of methanolic body tissue extract and fractionated gladius polysaccharide of squid ranged from 60 to 100 mg/ml. The results were discussed in the light of positive and negative control apart from the concentrations tested.


INTRODUCTION
The marine environment is a rich source of biologically active natural products of diverse structurally active natural products, many which have not been found in terrestrial sources.Antitumor, antiviral, antibacterial, anticoagulant, hemolytic, analgesic, cardio inhibitory, anticonvulsant, vasopressive and other active substances have been found in marine organisms and their surrounding environment.The marine organisms have had a major impact on the development of medical science.More recent studies on marine organisms have focused mainly on their application for the treatment of human diseases.Many marine chemicals often possess quite novel structures which lead to pronounced biologically activity and novel pharmacology (Lei and Zhou, 2001).A number of discovery efforts have yielded several bioactive metabolites which have been successfully developed by the pharmaceutical industry (Kong et al., 1994).From 1969 to 1999, approximately 300 patents on bioactive marine natural products were issued.From humble beginning, the number of compounds isolated from various marine organisms has virtually soared and now exceeds 10,000 (Marin Lit, 2002) with 100s of new compounds still being discover every year (Faulkner, 2000(Faulkner, , 2002)).
Molluscs are widely distributed throughout the world and have many representatives in the marine and *Corresponding author: E-mail: barwinrobin@gmail.com.Tel: +917598638767.Fax: 914144243555.estuarine ecosystems.Among the molluscs, cephalopods are very good sources of bioactive compounds.The fluid from the ink sac is found to have antibiotic effect and a pigment from the ink sac of the cuttlefish has been used in medicine, especially in homeopathy.Apart from the ink, the cuttlebone is also widely used as a home -made remedy for ear ache and skin diseases in India and China.In most of the publications concerning antimicrobial activity in molluscs, either single body components alone, like haemolymph and egg masses, or extracts of whole bodies have been tested for activity (Kubota et al., 1985).The presence of antimicrobial activity in molluscs has been reported from the mucus of the giant snail Achantia folica (Yamazaki, 1993), from the egg mass and purple fluid of the sea hare Aplysia kurodai and the body wall of the sea hare Dolabella auricularia (Iijima et al., 2003) and body tissue of six species of cephalopods (Ramasamy et al., 2011).The antimicrobial activity of polysaccharides extracted from cephalopods such as Sepia aculeata and Sepia brevimana and heparin and heparin -like glycosaminoglycans (GAGs) from the cephalopod Euprymna berryi was reported against the human pathogenic microorganism (Shanmugam et al., 2008a;Shanmugam et al., 2008b).Further, these compounds are being extracted not only from the whole animal but also from the different body parts including the skeleton (internal shell of cephalopods), which showed many pharmacological properties and hence medicinal value (Shanmugam et al., 2008a).
Scientific interest for cephalopods is increased over the last century for, at least a couple of reason: i) their value as experimental animals for biomedical and behavioral research (Hochner, 2008;Castellanos-Martinez and Gestal, 2013); ii) their position in the world marked as a major fishery resource (Boyle and Redhouse, 2005;Castellanos-Martinez and Gestal, 2013).The present study has attempted to focus on the attention on the study of antimicrobial activity shown by the mehtanolic extract of body tissue and fractionated gladius polysaccharide from squid gladius from two squid species (L.duvauceli and D. sibogae).

Sample collection and identification
Two species of squid such as L. duvauceli and D. Sibogae, were freshly collected from Cuddalore (Lat.11°42'N;Long.79°46'E) and Mudaslodai (Lat.11°29'N; Long.79°46'E) landing centre which is situated south east coast of India.The studies carried out by Shanmugam et al. (2002) have been of considerable help on developing the identification keys and description which in most cases have also been corroborated with examination of actual specimen.

Preparation of methanoloic extracts of body tissue
Squids were brought to laboratory; body tissues were removed, cut into small pieces and homogenized (REMI, RQ-127 A) and extracted with methanol (MeOH) at room temperature for 24-48 h (Ely et al., 2004).Then, the methanolic extract was centrifuged to collect the supernatant and concentrated under vacuum in a rotary evaporator (LARK, Model: VC-100A) at low temperature.The crude methanolic extract was assayed for antibacterial and antifungal activities using standard disc diffusion method.

Extraction of polysaccharide from squid gladius
The polysaccharide extract were obtained from the squid of L. duvauceli and D. sibogae by following the method of Okutani and Morikawa (1978).The air-dried shell powder was pulverized and washed with acetone.The powder was extracted with hot 10 mM EDTA solution and filtered (whatman No.1) with hyflosuper cel.Then, saturated barium hydroxide solution was added to the filtrate and allowed to stand over night.Then, the precipitate was filtered on a filter paper (Whatman.No.1) with hyflosuper cel and washed with distilled water.The precipitated was dissolved in 10 mM EDTA solution and was dialyzed against deionised water using 8 KDa cutoff dialysis membrane.The dialyzate solution present in the dialysis membrane was then freeze-dried and a pure white colored powder was obtained.This lyophilized powder was used for the antibacterial and antifungal activity.

Purification of polysaccharide using ion exchange resin
The crude polysaccharide was fractionated by ion -exchange chromatography on a column (2.5x15 cm) of Amberlite IRA -900 forms in aqueous solution of 1% polysaccharide and was applied to the column and washed initially with water.Then, the sample was recovered by stepwise elution with 0.4 M NaCl and 0.8 M NaCl at the flow rate of 1 ml per minute.Both elutes were combined, dialysed and lyophilized (Nishino et al., 1989).

Gel chromatography
Gel filtration was done by using Sephadex G-50 (Sigma).Purified polysaccharide sample (Approximately 3g /10 ml 0.2 M-NaCl, was applied to the column; the eluting medium was 0.2 M-NaCl and the temperature was 4°C at a flow rate of 60 ml/h.Fractions (15 ml) were collected and the active fractions were pooled then dialyzed against distilled water and freeze-dried (Laurent et al., 1978).

Estimation of sugar content
Sugar (in crude) content was determined by phenol -sulphuric acid method (Dubois et al., 1956).

Elementary analysis
The element of C, H and N content was analyzed using elemental analyzer, Elementer Vario EIII, Carlo Erba-1180.

Bacterial and fungal strains
Nine bacterial (Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Vibrio cholerae, Vibio parahaemolyticus, Staphylococcus aerues, Pseudomonas aeruginosa, Salmonella typhii and Shigella sp.) and four fungal (Candida sp., Rhizopus sp., Aspergillus flavus and Aspergillus fumigatus) strains were used for studying the antibacterial and antifungal activities of methanolic extract and fractionated gladius polysaccharide.These strains isolated from the HIV patients, were obtained from Raja Muthyiah Medical College Hospital, Annamalai University, Tamil Nadu, India.These are all human pathogens that have developed some resistance to common antibiotics particularly in the in the clinical environment.

Inoculum preparation for bacteria
Nutrient broth was prepared and sterilized in an autoclave at 15 lbs pressure for 15 min.All the nine bacterial strains were individually inoculated in the sterilized nutrient broth and incubated at 37°C for 24 h.Mueller Hinton Agar (MHA, Himedia) was prepared, sterilized in an autoclave at 15lbs pressure for 15 min and poured into sterile petridishes and incubated at 37°C for 24 h.The 24 h old bacterial broth cultures were inoculated in the Petri dishes by using a sterile cotton swab.

Inoculum preparation for fungi
Czapek dox (Hi-media) broth was prepared and sterilized in autoclave at 15 lbs pressure for 15 min.Four fungal strains were inoculated in the broth and incubated at 37°C for 72 h.The sterilized Czapek dox agar was poured into sterile petridishes and incubated at 37°C for three days.The 72 h old fungal broth cultures were inoculated in the petridishes using a sterile cotton swab.

Antimicrobial assay (disc diffusion method)
In vitro antibacterial and antifungal activity was determined following the method of El-Masry et al. (2000).Briefly, a suspension of each tested microorganism was carefully mixed in the tube containing bacterial and fungal inoculums and media for bacterial and fungal were plated separately, and the respective strains were cotton swabbed on Petri dishes.Sterile antimicrobial disc (Himedia) was impregnated with 50 μl of crude methanolic extract and fractionated gladius polysaccharide of the four concentrations tested.Positive control discs contained 50 μl of tetracycline (1 mg/ml) and negative control 50 μl of methanol and water.The stocks for methanolic extracts and fractionated polysaccharide were prepared in the concentration of 100 mg/ml.These impregnated discs were allowed to dry at laminar air flow chamber for 3 h, and were placed at the respective bacterial and fungal plates and incubated at 37°C for 24 h for bacteria and 72 h for fungi.The diameter (mm) of the growth inhibition halos produced by the methanolic extracts and fractionated polysaccharide of squids were examined.Result was calculated by measuring the zone of inhibition in millimeters.All the tests were performed in triplicates.

Determination of the minimum inhibitory concentration (MIC)
The methanolic extract and fractionated gladius polysaccharide of squid which showed significant antimicrobial activity was selected for the determination of MIC followed by the method of Rajendran and Ramakrishnan (2009).A stock solution of 100 mg/ml was prepared and was serially diluted to obtain various ranges of concentrations between 20 and 100 mg/ml.0.5 ml of each of the dilutions of different concentrations was transferred into sterile test tube containing 2.0ml of nutrient broth.To the test tubes, 0.5 ml of test organism previously adjusted to a concentration of 105 cells/ml was then introduced.A set of test tubes containing broth alone was used as control.All the test tubes and control were then incubated at 37°C for 24 h.After the period of incubation, the tube containing the least concentration of extract showing no visible sign of growth was taken as the minimum inhibitory concentration.

Statistical analysis
Data on the inhibitory effect of methanolic extracts and fractionated gladius polysaccharide of squid was analyzed by one-way analysis of variance (ANOVA) using SPSS-16 version software followed by Duncan's multiple range test (DMRT).P values ≤0.05 were considered as significant.

RESULTS
Sugar content of fractionated gladius polysaccharide showed 42.7 and 48.1% in L. duvauceli and D. sibogae respectively.At the same time, chemical analysis that is C, H and N content of fractionated gladius polysaccharide showed 29.6, 6.3 and 1.2% and 31.8,5.6 and 1.8% in L. duvauceli and D. sibogae respectively.
The results of antibacterial activity are presented in Table 1.The highest activities of 10 mm (inhibition zone) were recorded in 100% against K. pneumoniae and E. coli in D. sibogae and 5 mm against P. aeruginosa in L. duvauceli of fractionated squid polysaccharides.At the same time, the methanolic extracts showed the highest inhibition zone of 10 mm observed against E. coli in D. sibogae extract and against V. cholerae in L. duvauceli extract.The lowest inhibition zone of 4 mm was observed against S. typhii and K. pnemoniae in L. duvauceli and D. sibogae extract.
In 75%, maximum activities of 7 mm were observed against S. aureus, K. pneumoniae and E. coli in D. sibogae and 4 mm against S. typhii in L. duvauceli fractionated polysaccharide.The methanolic extract showed the maximum activity of 7 mm against Shigella sp. in L. duvauceli extract and 8 mm against S. typhii in D. sibogae extract.The minimum activity of 4 mm against K. pneumonia and S. aureus and E. coli in L. duvauceli and D. sibogae extract respectively was observed.
In 50%, 6 and 3 mm inhibition zone were recorded against K. pneumonoiae and E. coli and P. aeruginosa and V. parahemolyticus in D. sibogae and L. duvauceli respectively.Whereas the maximum activity of 6 mm was noticed against V. cholerae in L. duvauceli methanolic extract and 4 mm against V. parahemolyticus in D. sibogae methanolic extract.The minimum activity of 3 mm was recorded against Shigella sp. and V. parahemolyticus in both L. duvauceli and D. sibogae methanolic extracts.
In L. duvauceli (25%), 3 mm inhibition zone was observed against V. parahemolyticus, P. aeruginosa and E. coli.The activities were completely absent against B. subtilis and V. cholerae.In D. sibogae, it was 5 mm against B. subtilis, P. aeruginosa, S. aureus and E. coli, and 3 mm against Shigella sp., S. typhii and V. parahemolyticus and activity was absent against V. cholerae.In methanolic extracts it showed 3 mm inhibition zone against Shigella sp., V. parahemolyticus and S. aureus and P. aeruginosa in both L. duvauceli and D. sibogae extracts respectively.
In the case of control, no activity was recorded against any bacterial strain in the two species of squid studied.
All the results of antifungal activity are presented in Table 2.In the control, no activity was recorded for all the fungal strains in both L. duvauceli and D. sibogae.The maximum of 12 mm inhibition zone were recorded against Rhizopus sp. and A. flavus in L. duvauceli and 5 mm against A. fumigatus in 100% concentration.In 75%, 7 and 8 mm inhibition zones were observed against Rhizopus sp. and A. flavus respectively and A. fumigatus recorded an inhibition zone of 4 mm only whereas 6 mm inhibition zone were recorded in 50% against Rhizopus sp., 4 mm against A. flavus and 3 mm against A. fumigatus.In 25%, 5 mm inhibition zone were observed against Rhizopus sp., 3 mm against A. flavus and A. fumigatus.At the same time, the activity were completely absent against Candida sp.
In D. sibogae, the highest activity was observed (10 mm) against A. fumigatus in 100% and 4 mm against Rhizopus sp.In 75%, 7 and 4 mm inhibition zones were recorded against A. fumigatus and Rhizopus sp. and A. fumigatus respectively.3 mm inhibition zone was recorded against A. fumigatus and Rhizopus sp. in 25%.The activity was absent against Candida sp. and A. flavus.But at the same time, the methanolic extracts showed no activity against all the fungal strains studied.

MIC of the active extract against the test organisms
The MIC of bacterial strains results are given in Table 3. MIC values of methanolic extracts of L. duvaucel against bacterial strains such as Shigella sp., S. typhii, V. parahaemolyticus, V. cholerae, and S. aureus were 100, 100, 100, 80 and 100 mg/ml respectively.In D. sibogae, the MIC for P. aeruginosa, S. typhii, V. parahaemolyticus, S. aureus and E. coli was recorded as 100, 80, 80, 80 and 100 mg/ml respectively whereas in fractionated polysaccharide of D. sibogae, the MIC for S. subtilis, Shigella sp.,S.typhii, V. parahaemolyticus, K. pneumonia and E. coli was recorded as 80, 100, 100, 100, 80 and 80mg/ml respectively.At the same time in L. duvauceli, the MIC for E. coli noticed was 100 mg/ml.
The MIC of fungal strains results are given in Table 4.In fractionated polysaccharide, the MIC values for fungal strains such as A. flavus and Rhizopus sp.Were 80 and 100 mg/ml in L. duvauceli and 60 and 100 mg/ml in D. sibogae respectively.

DISCUSSION
Our results clearly show (Tables 1, 2, 3 and 4) that the methanolic and fractionated polysaccharide extract exhibited appreciable antimicrobial activity against human pathogenic bacteria and fungi.The usage of antibiotic disc susceptibility tests or disc-diffusion assays has the ability to rapidly identify active metabolites and therefore is particularly useful in the initial screening for antimicrobial activity and as the means for following activity during chemical purification (Gunthorpe and Cameron, 1987).
Antibacterial activity varies with the bioactive compounds of different species and pathogenic bacterial strains.Diluting extract usually weakens their antimicrobial activity.For the first time attempts to study the antimicrobial activity in marine organisms were initiated around 1950s (Jensen et al., 1996).Since that time, a large number of marine organisms from a broad range of phyla have been screened for their antimicrobial activity (Rinhart et al., 1981).Potent antibacterial activity in haemocytes and haemolymph has been detected in various molluscs (Anderson and Beaven, 2001).Antimicrobial peptides have been isolated and characterized from the haemocytes of Mytilus edulis (Charlet and Prem Anand).
In the present study, high antibacterial activity was found in K. pneumoniae, S. aureus and E. coli (D. sibogae) and highest antifungal activity was recorded against Rhizopus sp. and A. flavus (L.duvauceli).The activity of the gladius extracts was found to be high in 100% concentration than the other concentration.In general, as the concentration increased, activity also increased.Polysaccharide extracts of D. sibogae showed activity against 8 bacterial strains except V. cholerae but L. duvauceli showed activity against 7 bacterial strains except V. cholerae and B. subtilis.For antifungal activity, D. sibogae showed activity against 2 fungal strains (A.fumigatus and Rhizopus sp.) and L. duvauceli showed activity against 3 strains.Both extracts showed no activity against Candida sp.
In the study, wide spectral antibacterial and antifungal activity were recorded in almost all the extract which is the significant finding of this study.Acetone extracts of oyster Pteria chinensis, showed higher degree of inhibition zone against K. pneumoniae (5 mm), Streptococcus pneumoniae (4 mm), Serratia marcescens (4 mm) and Proteus mirabilis (4 mm) (Yang et al., 2005).Escapin isolated from sea hare Aplysia californica, was most sensitive to escapin, with a minimum inhibitory concentration (MIC) of 0.25g/ml.The pathogenic species S. aureus and P. aeruginosa were the next most sensitive to escapin, with MIC values of 0.31 g/ml.Bacillus strains showed the highest resistance to escapin, with MIC of 2.5 g/ml (Vivek et al., 2002).
The substances extracted from squid (L.duvauceli) ink tested for antibacterial activity, showed large inhibition zones against E. coli followed by Salmonella spp., V. cholerae and Staphylococcus spp.(Patterson and Murugan, 2000).The moderate antibacterial and antifungal activity were also reported from the extracts of various bivalve molluscs (Prem Anand and Patterson Edward, 2002) and broad spectrum of antibacterial activity has been reported for aqueous ink extracts of the cephalopods L. duvauceli and S. pharaonis against nine human pathogens (Patterson and Murugan, 2000).Antibacterial activity of the S. Pharaonis ink showed maximum activity of 9 mm for S. epidermidis and all other pathogens (P.arruginosa, E. coli, Citro bactor and K. pneumonia) showed lower activity (Nithiya et al., 2011).
A peptide (containing 12 cysteine of about 6.2Kda MW) from a blood mussel M. edulis showed antifungal activity against N. crassa (Anderson and Beaven, 2001); "Haminol-A" from the Hawaiian opisthobranch mollusc, Philliopsis speciosa showed 8 mm inhibition zone at /ml concentration against Saccharomyces cerevisae and no activity at all against C. albicans; "dolabelanin-B 2 " from the sea hare D. auricularai showed antifungal activity (Adam and Charis, 2004).The secondary metabolites Lissoclinolide isolated from ascidians possesses antimicrobial activity (Jayaseeli et al., 2001).Likewise in the present investigation also, the polysaccharide extracted showed potent antifungal activity against pathogenic fungi.In 100% concentration, the highest inhibition zone of 12 mm was observed against K. pneumonia and S. aureus in methanolic extract of whole body tissue and S. aureus alone in EDTA extract from cuttlebone.The lowest inhibition zone of 8 mm was observed against V. parahaemolyticus and Salmonella sp. in the methanolic extract of whole body tissue and V. parahaemolyticus alone in EDTA extract from cuttlebone (Vairamani et al., 2012).
Only very few studies have been carried out on the antimicrobial activity of the internal shell of cephalopods.But many such studies are available for the extracts from the whole body tissue whose results could be compared with that of the present study.When studied, the antibacterial activity of Donax modesta, Circe scripta and G. pectinatum against nine pathogenic bacteria such as S. aureus, K. pneumoniae, S. typhii, E. coli, B. subtilis, P. vulgaris, P. mirabilis, V. cholerae and S. flexeri reported broad spectrum antibacterial activity for the water and heptane extracts (Jeyaseeli et al., 2001).50 and 100 µl concentration of methanolic extract from body tissue of S. brevimana extract showed maximum zone (17 and 19 mm respectively) of inhibition against gram negative bacteria K. pneumonia (Mohanraju et al., 2013).In 100% concentration, the highest inhibition zone of 17 mm was observed against E. coli in whole body parts of Octopus dolfusi, 15 mm against V. parahaemolyticus in Octopus aegina and the lowest inhibition zone of against S.
The fact that some of the polysaccharide extracts showed high antibacterial and antifungal activity against human pathogens indicates that polysaccharide is at least not solely responsible for the antimicrobial activity detected.

Conclusion
In the present study, squids were collected from the Mudasalodai and cuddalore landing centre, southeast coast of India and showed potential antimicrobial activity against human pathogenic bacterial and fungal strains.Conceptually, it is clear that the marine ecosystem offers a huge potential as a natural based bioactive compounds.Fractionated products isolated from marine organisms have served as a source of drugs and starting material for the synthesis of useful drugs.In addition, because of the differences in the environmental conditions, marine organisms can evolve new or unusual biochemical entity having biological activity.So, it is believed that the studies of new and unique compounds derived from the marine organisms well continued to increase our basic knowledge with respect to pharmacology and medicine.We found potent antibacterial and antifungal activity against human pathogenic strains.This finding is very significant and may pave way for the discovery of new potent drugs against these dangerous pathogens.

Table 1 .
Antibacterial activity of methanolic extract and fractionated polysaccharides of squids.

Table 2 .
Antifungal activity of methanolic extract and fractionated polysaccharides of squids

Table 3 .
MIC of methanolic extracts and fractionated polysaccharides of squids against clinically isolated human pathogens.

Table 4 .
MIC of methanolic extracts and fractionated polysaccharides of squids against clinically isolated human pathogens.