Bioactive Potential of Some Intertidal Molluscs Collected from Mumbai Coast,West Coast of India
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Asian Pacific Journal of Tropical Biomedicine (2012)S1060-S1063 S1060 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Biomedicine journal homepage:www.elsevier.com/locate/apjtb Document heading doi:10.1016/S2221-1691(12)60361-X 襃 2012 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved. Bioactive potential of some intertidal molluscs collected from Mumbai coast,West coast of India Atul G. Babar1*, Anant Pande1, 2, Balasaheb G. Kulkarni1 1Department of Zoology, The Institute of Science, 15 Madame Cama Road, Mumbai- 400032, Maharashtra, India 2Department of Animal Ecology and Conservation Biology, Wildlife Institute of India, Chandrabani, Dehradun-248001, Uttarakhand, India ARTICLE INFO ABSTRACT Article history: Objective: To identify the bioactive potential of crude methanolic extracts oMethods:f five gastropods Received 11 June 2012 and one bivalve collected from the west coast of India (Mumbai coast). Disc- Received in revised from 5 July 2012 diffusion assay and micro-dilution techniquResults:e were u sed to test the bioactive potential of these Accepted 7 August 2012 molluscs against six pathogenic bacteria. The crude methanolic extracts of all the Available online 28 August 2012 molluscs showed significant activity against one or more bacteria tested. –Minimum inhibitory concentration of crude methanolic extrHemifususacts was f opugilinusund in the ranNeritage of 0 .sp10 0.35 mg/mL against human pathogenic bacterial strains. Hemifusus and pugilinus. extracts were most effective showing actVibrioivity a choleraegainst a ll bacterial strains. extract Gafrariumexhibited Keywords: divaricatumgood activity against contrary to earlier reportsEscherichia. Methanoli ccoli extract Streptococcusof demonstrated considerable activity against both and Antibacterial pyogenes. Trochus radiatus Escherichia extract was inactive against all the bacteria tested except I coli Euchelus asper Klebsiella pneumoniae Pseudomonas ntertidal molluscs . extract was active against and M aeruginosa Conclusions: inimum inhibitory concentration . All the molluscs screened exhibited potential activity against one or M umbai coast more bacterial strain. Further work is in progress to isolate the active constituents of the extracts. [2,3] 1. Introduction to natural products isolated from marine molluscs . The majority of research on natural products from the phylum Mollusca has been focused on primarily soft- The diversity of the ocean has been the source of unique bodied or shell-less molluscs, particularly nudibranchs chemical compounds, which holds tremendous pharmaceutical and opisthobranchs[4,5]. However some studies have also potential. In the period from 1969 to 1999 about 300 patents on been reported biological activity from shelled molluscs[6,7]; natural products derived from marine organisms have been contrary to the general assumption that presence of a shell issued[1]. Most of these studies have restricted themselves to provides all the required protection from predation[5]. Work sponges and other soft-bodied organisms for the search of has also been done on the antimicrobial activity of egg masses bioactive compounds. However, the vast variety of molluscs of the molluscs[8]. However research investigations on the in the marine environment provides an enormous resource for antimicrobial potential of the marine molluscs commonly natural products research[2]. found on Indian coast are rare. There have been a few studies specifically dedicated The intertidal area off the coast of Mumbai is highly polluted due to continuous exposure to domestic sewage. Yet it *Corresponding author: Atul G. Babar.Department of Zoology, The Institute of boasts of a high diversity of molluscs, especially gastropods Science, 15 Madame Cama Ro Atul G. Babar Department of Zoology, The Institute of and sponges. Therefore, it is rational to search for novel Science, 15 Madame Cama Road, Mumbai- 400032, Maharashtra, India. antimicrobial agents in this marine environment, as these Tel: +91-9029319382 benthic organisms are living in a soup of microorganisms[9]. Fax: +912222047962 E-mail: [email protected]; [email protected] During present investigation the methanolic extracts of five Atul G. Babar et al./Asian Paicfic Journal of Tropical Biomedicine (2012)S1060-S1063 S1061 viz. Nerita Euchelus asper Hemifusus in vitro pugilinusgastropods Trochus sp., radiatus (Gmelin), Bursa The results of the antibacterial assay of the marine tuberculata (Born), Gafrarium (Gmelin) divaricatumand organisms are summarized in Table 1. Distinct antibacterial (Brodrip) and one bivalve activity was obsNeritaerved against H.all pugilinusthe pathogenic bacteria. (Gmelin) were screened for bioactivity against clinical isolates The extracts of sp. and exhibited Neritabroad of six bacteria. spectrum activity against all the microorganisms tested. sp. showed weH.ak pugilinus activity (7-10 mm) against all the tested 2. Materials and methods bacteriaV. w hcholerae.ile T. radiatusexhibited good activity (10-15 mm) against extract was inactive towards all 2.1. Sampling the bacteria tested except E. coli against which it was weakK.ly pneumoniaeactive. Crude extraP.ct aeruginosa.of E. asper was weakly acG.ti vdivaricatume against and The K.bi vpneumoniaealve P. All the samples were collected manually from the Marine aeruginosaextract was weakly active against E. coliand Drive rocky shore at low tide. The samples were brought to S. pyogenes bB.ut tuberculatademonstrated good activity against and the laboratory and immediately frozen at -20 曟. The shell E. coli, K. pneumoniae. P.de maeruginosa.onstrated weak activity against was cracked open using a tight grip plier; whole body of all and the animals was removed and subsequently washed with Table 2 shows the MICs of crude methanolic extracts anH.d autoclaved distilled water to remove any attached debris. pugilinusStreptomycin G.co ndivaricatumcentrations against tested bacteria. The opercula of all the gastropods were detached from their and eV.xt rcholeraeacts demonsE.tra colited lowest respective body and discarded. The reference specimen of MIC value of 0.1 mg/mL against and strains each species was deposited in the Department of Zoology, Neritarespectively. HiT.gh radiatusest MIC value of 0.35 mgS./m pyogenesL was shown bE.y Institute of Science. coli sp. and K. ex pneumoniaetract against P. aeruginosa and strains respectively. and 2.2. Extract preparation T. radiatus were inh iE.bi tcolied significantly by all extracts excE.ep aspert extract. whereas did not show any response to The whole body of each sample was cut into pieces, For each of the six bacterial strains, the MIC of Streptomycin homogenised in a blender with methanol and kept on shaker was alsoK. e spneumoniaetimated. It was found to be lowest (0.002 mg/mP.L) for 24 h. The supernatant was then filtered and concentrated aeruginosaagainst P. aeruginosa and highest (0.06 mg/mL) against under vacuum on a Rotary Evaporator at low temperature and . TV.h ucholeraes, was thE.e mcoli,ost rB.es isubtilisstant strain reduced pressure to get crude methanolic extract. S.fol lpyogenesowed by (0.005 mg/mL). anK.d pneumoniae (0.003 mg/mL) were equally resistant while 2.3. Antibacterial assay (0.002 mg/mL) strain was the least resistant strain used in the study. Antibacterial activity was tested against six bacterial strains Figure 1 compares the inhibition zones of different molluscs Escherichia coli Pseudomonas aeruginosa against each bacterial strain. It can be observed that crude viz. Klebsiella, pneumoniae , Streptococcus Nerita pyogenes, , Bacillus subtilis and Vibrio methanolic extract of B. s psubtilis. shows maxK.im pneumoniaeum activity cholera using standard agar disc-diffusion assays [10]. Each against pathogenic bacteria E. asperand B. tuberculata, compared to other strains. Extracts of , microorganism was inoculated on the8 surface o8f a nutrient agar H. pugilinus G. divaricatum plate at a concentration of 1.2 伊 10 -1.5 伊 10 CFU/mL using P. aeruginosa, and V. cholerae, s B.ho wsubtilis equally hiS.gh pyogenes activity a sterile glass spreader. Previously sterilized Whatman No. 1 against and filter paper discs (6 mm in diameter) were impregnated with 0.5 werNeritae found to be reH.sis tpugilinusant against all the exE.tra colicts e xcept those mg/mL solution of each crude extract and were placed on the of G. divaricatum sp. and . Against the extract surface of inoculated plate. Each plate also had a Streptomycin of exhibited maximum activity while all the disc (0.1 mg/mL) as a positive control and methanol disc as other eT.xt rradiatusacts were weakly active. LeaE.st acolicti vfollowede extrac t wE.as negative control. The plates were incubated at 37 曟 for 24 h. tasperhat of which inhibited only P. aeruginosa by K. All the assays were carried out in triplicates. The bioactivity of pneumoniae which was weakly active against and ( ) . the extracts was measured by calculating the diameter mm of 20.00 15.00 Nerita the growth inhibition halos. Zones of growth inhibition greater 10.00 S. pyogenes E.asper sp 5.00 B.tuberculata [11] 0.00 than 7 mm were considered susceptible to crude extracts . 20.00 T.radiatus 15.00 H. pugilinus 10.00 B. subtilis G. divaricatum 2.4. Determination of MIC 5.00 20.000.00 Streptomycin 15.00 10.00 P. aeruginosa 5.00 20.000.00 ( ) 15.00 Minimum Inhibitory Concentration MIC of crude extract was 10.00 K. pneumonia ( ) 5.00 determined for serially diluted concentrations 0.05-0.5 mg/mL 20.000.00 15.00 of crude methanolic extracts against each individual human Inhlbltion zone In mm 10.00 V. cholerae 5.00 0.00 pathogenic bacterial strain. Previously described disc diffusion 20.00 15.00 [12] 10.00 E. coli assay was used to calculate the MIC . Disc of Streptomycin of 5.00 concentration ranging from 0.001-0.1 mg/mL respectively were 0.00 used as a positive control and methanol was used as negative Figure 1. control. MIC of Streptomycin against each bacterial strain was Antibacterial activity of the molluscan extracts (0.5 mg/mL) also calculated by using the disc diffusion assay as described compared to the positive control of Streptomycin (0.1 mg/mL).