In vitro Antibiotic Bustle of Coral Reef Associated Gastropod, Drupa margariticola (Broderip, 1832) of Tuticorin Coastal Waters, Southeastern India
To test the antibacterial effect of the extracts of Drupa margariticola obtained using low to high polar solvents, like ethyl acetate, dichloromethane, acetone and methanol. Partial purification of the active crude extract was carried out using column chromatography employing a step gradient solvent system. A maximum inhibition of 7 mm against E. coli was shown by the 100% acetone column purified fractions of D. margariticola at a concentration of 0.125 mg. Minimum Inhibitory Concentration values were found to be lower for the 100% acetone fraction for pathogens, E. coli (0.05 mg), Klebsiella pnuemoniae (0.05 mg), Pseudomonas aerogenosa (0.07 mg) and Streptococcus pnuemoniae (0.07 mg). Thus 100% acetonated fraction of the extract of D. margariticola was considered as potent antibacterial compounds against some human pathogens. The antibacterial potential of the mollusc, Drupa maragariticola becomes a corner stone for the future development of novel biologically active compounds.
The oceans are the source of a large group of structurally unique natural products
that are mainly accumulated in bacteria, invertebrates such as sponges, tunicates
and bryozoans and also in molluscs. Research on bioactive compounds from marine
organisms has provided the broad and better support of marine natural products
research throughout the past quarter century. Serious attempts to tap the vast
potential of marine organisms as sources of bioactive metabolites that may be
directly utilized as drugs or serve as lead structures for drug development
started in late 1960s. Marine natural products chemistry is essentially a child
of the 1970s that developed rapidly during the 1980s and matured
in the last decade (Faulkner, 2005).
As a consequence of an increasing demand for the biodiversity in the screening
programmes seeking therapeutic drugs from natural products, there is now a greater
interest in marine organisms. There is a copious number of works pertaining
to the discovery of antibacterial agents from marine bacteria (Fenical
and Jensen, 1993; Kobayashi and Ishibashi, 1993),
seaweeds (Fuller, 1994; Gerwick,
1993), sponges (Ireland et al., 1993; Fusetani
et al., 1987; De Silva et al., 1992;
Longley et al., 1991), molluscs (Zhang
et al., 1994; Schmitz et al., 1993;
Wright, 1998; Fenical, 1997; Chellaram
and Edward, 2009) and ascidians (Davidson, 1993;
Rinehart et al., 1993; Sakai
et al., 1992). The cone shaped gastropod Drupa margariticola
is commonly occurring long the Tuticorin costal waters were chosen in an attempt
to test the antibacterial activity with the crude and column purified extracts.
Also, the Minimal Inhibitory Concentration (MIC) of the column-purified fractions
MATERIALS AND METHODS
Extraction of crude extracts: The samples were collected in the intertidal
region (using SCUBA diving) of the Tuticorin coastal waters (Lat 8°45 and
Long 78°13E) (Fig. 1) and immediately brought to
the laboratory. The animals were thoroughly washed with fresh water to remove
the salt and debris and air-dried. Approximately 20 g of the air dried sample
was taken and immersed separately in different solvents such as ethyl acetate,
acetone, dichloromethane and methanol and cold steeped at 18°C. The extract
from each solvent was filtered separately using Whatmann No.1 filter paper.
The filtrate was poured in previously weighed petri plates and evaporated to
dryness (Chellaram and Edward, 2009b; Becerro
et al., 1994; Riguera, 1997; Wright,
1998) and the dried extract was used for the antibacterial assay.
Antibacterial assay: To test the antibacterial effect of the extracts
obtained using different solvents, E. coli, Shigella dysentriae,
Stphylococcus epidermidis, S. areus, Klebsiella pneumoniae,
Pseudomonas aerogenosa, Salmonella typhimurium, S. paratyphi,
Vibrio cholerae, Streptococcus pneumoniae, S. faecalis,
Bacillus subtilis, B. cereus, Enterococcus aerogenosa and
Citrobacter sp. were used as a test strains. All the test strains were cultured
in Nutrient Broth (NB) and the 12-18 h old cultures were used for the tests.
The antibacterial assay was performed by using of the standard Nathans
Agar Well Diffusion (NAWD) technique (Nathan et al.,
1978) against the test strains on 50% Nutrients Agar (NA) in petridishes
with drilled wells of 6 mm diameter. The 0.2 and 0.4 mg of the dried extract
in 50 μL Dimethyl Sulfoxide (DMSO) was loaded onto each well. The well
at the center served as the control (without the extract). After 22-24 h of
incubation at room temperature, the susceptibility of the test organisms was
determined by measuring the radius of the zone of inhibition around each well
which is the distance between the border of the well and the edge to where the
test strains are completely inhibited.
Partial purification of the active crude extract: Partial purification
of the active crude extract was carried out following the method of Wright
(1998). After primary screening, the extract showing activity obtained from
acetone was fractionated using normal phase silica gel (200-400 mesh, LOBA CHEMIE,
Mumbai) column chromatography employing a step gradient solvent system from
low to high polarity. The step gradient protocol used was: 100% hexane; 80%
hexane: 20% acetone; 60% hexane: 40% acetone; 40% hexane: 60% acetone; 20% hexane:
80% acetone; 100% acetone; 80% acetone: 20% methanol; 60% acetone: 40% methanol;
40% acetone: 60% methanol; 20% acetone: 80% methanol and finally 100% methanol.
Each of the dried fractions was dissolved in 5 μL DMSO and were again tested
for antibacterial activity. After 24 h of incubation, the susceptibility of
the test organisms was determined by measuring the radius of the zone of inhibition
around each well.
Determination of Minimal Inhibitory Concentration (MIC): The Minimal Inhibitory Concentration (MIC) of the active column-purified fractions were determined by serially diluting the active column purified fractions so that concentrations of 250, 200, 150, 100 and 50 μg in 50 μL DMSO were loaded in to each well for the individual pathogenic strains that were found to be highly susceptible. The work was carried out at Suganthi Devadason Marine Research Institute, Tuticorin and Veltech Multitech Dr. RR Dr. SR Engineering College, Chennai. India, during April 2008 to April 2009 and one of the author is professionally trained international certified advanced level SCUBA diver.
Antibacterial Activity of crude extracts: Out of the 4 solvents used
for the extraction of the gastropod, Drupa margariticola the extract
with acetone was found to produce a distinct zone of inhibition 8 and 7 mm against
Salmonella typhimurium, Vibrio cholerae, Bacillus subtilis
and Staphylococcus epidermidis, Enterobacter earogenes and Citrobacter
sp., respectively at a concentration of 0.2 mg. Similarly the methanol extract
was also able to produce zone of 8 mm against Vibrio cholerae at the
concentration of 0.2 mg. However, the ethyl acetate was able to produce zone
of 7 mm against V. cholerae. On the other hand, methanol crude extract
was able to produce a zone of 2 mm against Pseudomonas aerogenosa and
Salmonella paratyphi at the same concentration (Table 1).
Antibacterial activity of the column purified extracts of Drupa margariticola
against human pathogens: Table 2 shows the effect of the
column purified extracts of D. margariticol against human pathogens.
A maximum inhibition of 7 mm against E. coli was shown by 100% acetone
column purified fractions at concentration of 0.125 mg. Similarly, the 100%
acetone fraction of column purified fraction of D. mrgariticola produced
a 6 mm against Staphylococcus epidermidis, Klebsiella pneumoniae,
Pseudomonas aerogenes and Vibrio cholerae at the same concentration.
However, somewhat lesser inhibition of zone was shown by combination of 80%
acetone and 20% hexane followed by 60% acetone and 50% hexane fractions. On
the other hand, 100% hexane; 80:20 hexane: acetone; 20:80 acetone: methanol
and 100% methanol fractions showed only trace inhibition.
Minimal Inhibitory Concentration (MIC): The Minimal Inhibitory Concentration
(MIC) values were found to be lower for the 100% acetone column purified fractions
for the pathogens, E. coli (0.05 mg), Klebsiella pneumoniae (0.05
mg), Pseudomonas aerogenes (0.07 mg), Streptococcus pneumoniae (0.07
mg) (Table 3).
activity of gastropod Drupa margariticola against human pathogens
|Note: A: Acetone, EA: Ethyl acetate, DCM: Dichloromethane
and ME: Methanol
activity of the column purified fractions of D. margariticola against
|Note: A: Acetone, H: Hexane, ME: Methanol and T: Trace
of the column purified fractions of Drupa margariticola against
|A: Acetone, H: Hexane and ME: Methanol
The sea has immense biomedical potential which can be exploited not only as
a source of drugs for treatment of disease but also of new and novel structures
with useful biological activities. In the past 25 years, marine organisms- mollusks,
algae, plants and microbes have provided key structures and compounds that proved
their potential in several fields, particularly as new therapeutic agents for
a variety of diseases. The interest in the field is reflected by the number
of scientific publications, the variety of new structures and wide scope of
organisms investigated (Faulkner, 1996).
In the present study, pronounced inhibition was conferred by the acetone extracts
of D. margariticola against the 15 dreadful human bacterial pathogens.
Earlier work performed on the antibacterial activity of the winged mollusc,
Pteria chinensis reported that out of the 6 solvents used, the extract
obtained from acetone and chloroform exhibited higher antibacterial activity
against human pathogens which stands by the present work (Chellaram
et al., 2004). Also, there is finding to report that the acetone
extracts of different seaweeds showed antibacterial properties against human
pathogens (Sureshkumar et al., 2002). On the
contrary, Anand and Edward (2002) reported that the crude
methanol extracts of Cypraea errones exhibited higher antibacterial and
antifungal activity. In this study, extracts from other solvents tested showed
only moderate type of inhibition suggesting that these extracts may not possess
potent antibacterial compounds. But a work executed on the in vitro antimicrobial
susceptibility test of the red, green and brown macroalgae showed that the methanolic
extracts were efficient in their action (Gonzalez del Val
et al., 2001). Similarly, Anand et al. (1997)
have also reported a broad spectral activity for the methanolic extract of Rapana
rapiformis egg capsules against nine pathogenic bacteria.
The acetone extract of the D. margariticola which was found to possess
higher antibacterial activity was hence chosen to localize the active component
through column purification. Yet again the 100% acetone column purified fractions
were found to possess utmost antibacterial activity. The inhibition zone of
7 mm was shown by the 100% acetone column purified fractions against E. coli,
at a concentration of 0.125 mg which is much lesser than the concentration of
the crude extract (0.2 mg). Chellaram et al. (2004)
have reported that the acetone fractions of mollusk, Pteria chinensis exhibited
broad spectral antibacterial activity that substantiates the present finding.
But on the contrast, the crude extract of Chicoreus virgineus, after
antibacterial assay-guided elution, showed activity only in 100% methanol fraction
(Ramasamy and Murugan, 2003). The Minimal Inhibitory
Concentration of D. margariticola was found to be lower for the 100%
acetone phases (0.05 mg) for E. coli. However, MIC values of 20 μg
mL-1 were recorded for the metabolites of soft corals, Caldiella
sp. and Sinularia sp. for human pathogenic bacteria (Radhika
et al., 2003). The study by Kelman et al.
(2001) has focused in determining the MIC values of the pure compounds obtained
from the sponge, Amphimedon viridis for selected marine bacteria and
the authors have estimated that the values are greater than 250 μg. This
finding suggests that since antibacterial activity was more pronounced at the
100% acetone phase with very minimal concentration against dead full human pathogens.
Authors thank sincerely the Director, Suganthi Devadason Marine Research Institute, Tuticorin, for the financial and laboratory support and Chairman and Director, Veltech Multitech Dr. RR Dr. SR Engineering College, Chennai, for their unremitting encouragement.
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