Exploration of Potential Antimicrobial Activity of Sea Star Astropecten indicus

The antimicrobial activity of crude tissue sample of sea star Astropecten indicus collected from Mudasal Odai, southeast coast of India was evaluated with the testing human microbial pathogens viz., Escherichia coli, Klebsiella pneumonia, K. oxytoca, Staphylococcus aureus, Streptococcus sp., Pseudomonas aeruginosa, Salmonella paratyphi and S. typhi using solvent system methanol and ethyl acetate at the concentrations of 250, 500 and 1000μl by well diffusion method. The crude methanol extracts showed more active exhibiting broad spectrum of antimicrobial activity than the crude ethyl acetate extracts. The maximum inhibiting zone of 13.44±0.20 was observed for bacteria Pseudomonas aeruginosa in the crude obtained from methanol followed by ethyl acetate 11.26±0.09 against Klebsiella pneumonia at the concentration of 1000μl. Moderate activity was found for all the tested strains at 500μl concentration of both the solvents. Bacterial strains K. oxytoca, Staphylococcus aureus has showed no activity at 250μl for methanol and the zone of inhibition for the tested clinical isolates was lesser than the zone of antibiotics used and no zone was found for the negative control. The present work reveals that, sea star A. indicus has potential antimicrobial activity.


INTRODUCTION
Ocean has plenty of organisms which are evolved with potential secondary metabolites being used as medicine (Devi et al., 2011;Rajeev Kumar Jha and Xu Zi-rong, 2004).Sponges, ascidians, bryozoans and molluscs are largely used for the production of novel compounds in a larger proportion (Proksch et al., 2002) but only less than 1% of the isolated compounds examined so far for pharmacological activities from the marine organisms (Fusetani, 2000).The multi resistant nature of pathogens to antibiotic is the serious threat and has stimulated search for novel antimicrobial agents from various natural sources (Laila Abubakar et al., 2012).During the last decade, there has been an increase in research on marine crustaceans, molluscs and echinoderms, particularly interest on their secondary metabolites with desirable antimicrobial properties (Casas et al., 2010;Haug et al., 2002).
Unexpectedly, echinoderms appear as untapped source in the pursuit of the identification of new and useful products (Petzelt, 2005).Sea stars are benthic free living echinoderm has evolved with rich sources of bioactive metabolites such as steroidal glycosides, steroids, anthraquinones, alkaloids, glycolipids and phospholipids de Marino et al., 1997;Palagiano et al., 1995;Pathirana and Andersen 1986).Especially steroidal glycosides and related compounds are predominant metabolites in sea stars and have a broad variety of biological activities such as cytotoxic, hemolytic, ichthyotoxic, repellent, antineoplastic, antimicrobial, antifungal, antiviral and anti-inflammatory (Andersson et al., 1989;Chludil et al., 2002;Ivanchina et al., 2000;Prokofeva et al., 2003;Wang et al., 2002;Wang et al., 2004).Imbricatine from the sea star Dermasterias imbricata is the first benzyltetrahydroisoquinolone alkaloid from a non-plant source and shows in the NCI human cellline screen (Carte, 1996).Cerebrosides and gangliosides have been reported to exhibit various pharmacological effects including antitumour and neuritogenic activities (Endo et al., 1986;Higuchi et al., 1993;Hirsch and Kashman 1989).Saponins are widespread in sea stars (Iorizzi et al., 2001).
Astropecten indicus are paxillosidan sea stars generally found within the sediments of intertidal and subtitdal regions where they voraciously feed on infaunal molluscs.They widely distributed in the Indian waters and reproduces enormously.India has rich resources of asteroids in the Gulf of Mannar and Palk Bay region but they are not being utilized for any other purpose.Only few studies have been made on the some of the sea stars of Indian waters regarding pharmacological potential (Kanagarajan et al., 2008) and most of the studies are restricted to only taxonomy (Karuppaiyan, 2007).By considering the above mentioned factors, the present work was designed and carried out to assess the potential antimicrobial activity on human pathogens.

Sample collection and preparation of extracts
Live samples of A. indicus were collected from the sea Bay of Bengal at the fish landing center Maudasal Odai (Lat.11 o 29′N; Long.79 o 46′E) southeast coast of India during the month of august 2011.Freshly collected samples were immediately washed to remove adhered mud and other particles and brought to the laboratory in a frozen condition.The extraction procedure was followed by modified method of Chellaram et al., (2004).50gms of chopped tissues samples were placed on the solvent methanol and acetone separately in the ratio of 1:3 (w/v) for 24 hrs at normal room temperature, then extracts were filtered by Whatman filter paper No.1 and the solvents were concentrated by rotary evaporator (VC100A Lark Rotavapor® at 30ºC) under reduced pressure and temperature, the resultant residues were stored at 4ºC for further analysis.

Microorganisms used for screening
Details of human pathogenic strains used for screening were Escherichia coli, Klebsiella pneumonia, K. oxytoca, Staphylococcus aureus, Streptococcus sp., Pseudomonas aeruginosa, Salmonella paratyphi and S. typhi.All the pathogenic bacterial strains were obtained from Raja Muthaiah Medical College and Hospital, Annamalai University.

Antibacterial assay
The bioassay was carried out using the well diffusion method (Reinheimer et al., 1990).Muller Hinton agar plates are prepared by pouring 15ml of medium and allowed to solidify.The petri plates are swabbed with 24hrs old culture of the eight selected bacterial strains.Wells were loaded with 250, 500 and 1000µl of extracts prepared by dissolving 1mg of crude extracts in 1ml of distilled water.Streptomycin 400µl was used as positive control and negative control was prepared using distilled water.Plates were incubated for 24hrs at 37ºC.Zone inhibition was recorded in millimeters and three replicates were maintained.

RESULTS AND DISCUSSION
The radius of inhibition zone for crude extracts has shown in table 1&2.Results were expressed with mean and standard deviation by using Microsoft excel.The maximum inhibition zone 13.44±0.20 was observed for P. aeruginosa at the concentration of 1000µl of crude extraction obtained by methanol and minimum was observed 3.37±0.03for E. coli at the concentration of 250µl but no activity has been observed for K. oxytoca and S. aureus at the concentration of 250µl.
The results showed different activity for ethyl acetate extraction.It was higher 11.26±0.09against K. pneumonia at the concentration of 1000µl and minimum value was 1.13±0.06against S. aureus at 250µl.The control showed very good activity against P. aeruginosa in methanol extracts and against K. pneumonia in ethyl acetate extraction.There is no inhibition zone found for the negative control.The extracts of methanol showed higher antimicrobial activity almost for all the concentrations.
The crude methanol and ethyl acetate extracts of tissue samples of A. indicus showed high antimicrobial activity against pathogenic isolates of P. aeruginosa and K. pneumonia.Moderate activity also obtained for S. paratyphi, S. typhi, Streptococuus sp., and E. coli.Many enough literatures are supports the activity of sea stars extracts obtained from various geographical locations and many drug discovery projects have screened echinoderms against various human bacterial, fungal, viral and fish pathogens (Sri Kumaran et al., 2011).Prabha Devi et al. (2011) accounted that the body wall extract of sea star Pentaceraster affinis showed considerable activity against Shigella flexineri, Acinetobacter sp., and moderate activity against Sreptococcus pyogenes.They suggested that the compounds inhibited the activity of pathogen may be saponins and saponin like steroid derivatives.James (2010) reported that considerable toxicity is found in the echinoderms and the "crown of thorns" Acanthaster planci is highly toxic to human as well as fish predators.Sri Kumaran et al. (2011) also reported that the butanol extracts of Protoreaster lincki showed high activity against S. paratyphi and K. pneumonia.Methanolic extracts of P. regulus showed activity against K. oxytoca.They also stated that various extracts showed activity against fish and fungal pathogens.Kanagarajan et al. (2008) assessed the toxicity effect of Stellaster equestris and reported the presence of histamine like compounds which are toxic.The compound which is responsible for the respective activity may be of saponins and glycosides groups.Rinehart et al. (1981) has documented as about 43% of 83 unidentified California echinoderms have antimicrobial property.Yeon Jung Jung (2002) reported that the fractions obtained from Asterias amurensis and Asterina pectinifera showed strong activity against various human bacterial and fungal pathogens and also affirmed that the pre-treated fractions with protease showed stronger result than the positive control used.Rajiv Kumar Jha and Xu Zi-rong (2004) has reviewed various bioactive compounds from sea stars, sea urchin and sea cucumbers and reported that the major compounds in the sea stars are saponins, steroidal glycosides and alkaloids.Li et al. (2010) has reviewed many AMPs (antimicrobial peptides) from echinoderms which has strong antimicrobial activity against bacterial, fungal and viral pathogens.

CONCLUSION
Based on the assay conducted and surveyed literatures we concluded that the sea star Astropecten indicus has potential antimicrobial activity of many polar compounds particularly against P. aeruginosa and K. pneumonia.This study is suggesting that many promising compounds which have potential pharmaceutical values from these untapped sources to be evaluated for curing many diseases.A. indicus are underutilized for human usage and they can be explored for other pharmaceutical applications with sustainable conservation.

Table . 1
: Methanolic extracts of Astropecten indicus against human pathogen at various concentrations and the zone of inhibition (mm) with Mean and SD.

Table . 2
: Ethyl acetate extracts of Astropecten indicus against human pathogen at various concentrations and the zone of inhibition (mm) with Mean and SD.