Antibacterial Potential of Marine Ascidian Phallusia arabica Against Isolated Urinary Tract Infections Bacterial Pathogens

MATERIALS AND METHODS

Collection and preparation of samples: The ascidian Phallusia arabica (Chordata: Ascidiacea: Pleurogona: Pyuridae) were collected during the low tide of the intertidal area at Thoothukudi coast, Tamil Nadu, Southeast coast of India, between 20 and 23 August 2010. The collected samples were rinsed with sterile seawater to remove associated debris and salt. The samples were weighed (10 g) and preserved separately in methanol and ethyl acetate (1:2) and brought to the laboratory. Samples were then soaked in the above mentioned solvents for 48 h, the extracts were then obtained from the soaked samples by grinding, using pestle and mortar and filtering through Whatman No. 1 filter paper, the filtrate was centrifuged at 3000 rpm. The solvent was evaporated under reduced pressure using desiccator and the residue was weigh and dissolved in dimethyl formamide for using them for the antibacterial activity. All the urinary tract infection bacterial pathogen strains were obtained from Raja Muthiah Medical College, Annamalai University.

Antibacterial succeptiblility assay: Ascidian crude extract was tested for inhibition of bacterial growth against urinary tract infections bacterial pathogens. The extract of P. arabica was tested for antibacterial activity at two different concentrations by disc diffusion method (Avelin et al., 1991).

Minimum Inhibitory Concentration (MIC): Minimum inhibitory concentration was determined by the following procedure (Collins et al., 1995).

Minimum Bactericidal Concentration (MBC): Minimum bactericidal concentration was experimented after the MIC in freshly prepared agar plates, followed by standard method of Alade and Irobi (1993).

RESULTS AND DISCUSSION

In vitro antibacterial screenings of marine ascidians against selected clinical isolates were performed and the inhibition zones of the extract against the specific test organisms were given in Table 1. Maximum inhibition zone (12 mm) was observed against P. mirabilis in crude ethyl acetate extract (1 mg mL^-1 concentration) of P. arabica and the minimum inhibition zone (1.5 mm) was observed against P. aeruginosa in methanol extract (1 mg mL^-1 concentration). This view is consistent with the findings of Anand and Edward (2002) reported that ethyl acetate crude extract of ascidian exhibited strong antibacterial activity than methanol extract and Petroleum ether of all the strain tested. The methanol crude extract of P. Arabica, the range of inhibition of the clinical isolates of the bacteria varied from 1.5-6 mm was observed in both (500 μg mL^-1 and 1 mg mL^-1) concentration. This result is lesser than the findings of Hussain and Ananthan (2009), who reported that the crude methanol extract of Didemnum psammathodes, the range of inhibition of the bacteria varied from 2-15 mm with an average of 4.3 mm. On the other hand, Ramasamy and Murugan (2003) have reported that the crude methanol extract of Didemnum psammathodes, the range of inhibition of the bacteria varied from 6 and 10 mm with an average of 7.1 mm. The Ethyl acetate extract of T. clinids, the range varied between 1 and 8.5 mm with an average of 3.18 mm.

The crude ethyl acetate extract of P. arabica showed the maximum antibacterial activity in (1 mg mL^-1 concentration) against P. mirabilis, followed by E. coli, P. vulgaris, Klebsiella sp and P. aeruginosa and the minimum activity was noticed in all 500 μg mL^-1 concentration of the same extract. The crude methanol extract of P. arabica showed the maximum antibacterial activity in (1 mg mL^-1 concentration) against P. mirabilis, followed by P. vulgaris, E. coli, Klebsiella sp. and P. aeurginosa. There is no activity against all 500 μg mL^-1 concentration methanol extract (Table 1).

The minimum inhibitory concentrations (MICs) and minimum bactericidal concentration (MBCs) of the crude extracts were shown in Table 2. The range of MIC varied between 0.25-1.85 (500 μg mL^-1 and 1 mg mL^-1 concentrations) against all the bacterial strains used in this study and MBC ranges between 0.20-2.10 (500 μg mL^-1 and 1 mg mL^-1 concentrations) against all the bacterial strains. Ascidians are already reported for rich nitrogenous source with a wide range of biological activities (Biard et al., 1994). The range of MICs is highest in ethyl acetate extract 1.85 (1 mg mL^-1 concentration) and methanol extract 1.05 (1 mg mL^-1 concentration). Range of MBCs was high in ethyl acetate extract 2.10 (1 mg mL^-1 concentration) and methanol extract 1.15 (1 mg mL^-1 concentration). The range of MICs was low in the ethyl acetate extract 0.25 (500 μg mL^-1 concentration) and methanol 0.25 (1 mg mL^-1 concentration). The range of MBCs was low in ethyl acetate extract 0.20 (500 μg mL^-1 concentration) and methanol 0.60 (1 mg mL^-1 concentration). On the other hand, Natarajan et al., (2010) reported that the Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal Concentration (MBC) of the methanolic extract of Polyclinum madrasensis, MIC varied between 0.70-0.95 mg mL^-1 and MBC ranges between 0.85-1.10 mg mL^-1 against all the bacterial strain tested.

Table 1:	Antibacterial activity of Phallusia arabica
-: No activity was observed

Table 2:	MIC and MBC of different concentration crude extracts of Phallusia arabica
-: No activity was observed

Shobu and Pawlik (2007) reported that crude organic extracts of whole tunic and internal tissues of P. nigra contained vanadium metabolites (225 and 750 ppm dry mass, respectively) and were palatable to blue head wrasse, Thalassoma bifasciatum. Crude extracts also exhibited no antimicrobial effects against a panel of four marine bacteria (Vibrio parahaemolyticus, Vibrio harveyi, Leucothrix mucor and Deleya marina). Jaffarali et al. (2008) reported that the test of P. nigra exhibited a bactericidal effect, with the zone of inhibition ranging from 2.3 to 12.3 mm against all pathogens tested, as referred to the mantle body. This result more or less consistent to our results. Dowzicky and Park (2008) reported that UTI bacterial pathogens have exhibited decreased susceptibility rates to tigecycline over the years. Marine genus synthesizes active constituents which are used in traditional and complementary medicine (Anbu-Jeba-Sunilson et al., 2009). Antibacterial compounds from natural resources would be the alternative to overcome the resistance problem. Since, the present study indicated that the crude extract of P. arabica showed antibacterial activity which could be used in pharmacological research.

CONCLUSION

Present study deduce that the continuing and overwhelming contribution of ascidians metabolites to the development of new pharmaceuticals are clearly evident and need to be explored. After taking in to consideration the immense side effects of synthetic drugs, great attention has to be paid for the discovery of novel drugs from marine natural products.

ACKNOWLEDGMENT

Authors thank Prof. Dr. T. Balasubramanian, Dean, CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University and the Ministry of Earth Sciences (OASTC) New Delhi for financial assistance. We acknowledge Dr. H. Abdul Jaffar Ali, Assistant Professor, Department of Biotechnology, Islamiah College, Vaniyambadi for his help and advice.