Antibacterial Activity of Methanolic Extract of Whole Body Tissue and Ethylene Diamine Tetra Acetate Extract of Cuttlebone of Sepiella inermis (Orbigny, 1848)
A. Barwin Vino,
Marine animals have great potential for providing novel drug leads with novel mechanism of action. The aim of the present study was to evaluate the antimicrobial potency of methanolic extract of whole body tissue of Sepiella inermis and Ethylene Diamine Tetra Acetate (EDTA) extract of cuttlebone (polysaccharides) against ten human pathogens. S. inermis tissue was extracted with 100% methanol for about 48 h, centrifuged and supernatant was concentrated under vacuum in desiccator. The polysaccharide extract was obtained from the internal shell using 10 mM hot EDTA. The final product was used for assaying the antibacterial activity by disc diffusion method in different concentrations. In 100% concentration, the highest inhibition zone of 12 mm was observed against Klebsiella pneumoniae and Staphylococcus aureus in methanolic extract of whole body tissue and Staphylococcus aureus alone in EDTA extract from cuttlebone. In 75% concentration, methanolic extract showed highest activity of 9 mm against K. pneumoniae, Staphylococcus aureus and Staphylococcus pneumoniae whereas the EDTA extract showed highest activity 8 mm against Vibrio cholerae, Klebsiella pneumoniae and Vibrio alginolyticus. In 50% concentration, the maximum activity of 9 mm was recorded against Klebsiella pneumoniae and Staphylococcus aureus in methanolic extract; whereas maximum activity (11 mm) was recorded against E. coli in EDTA extract. In 25% concentration, the both maximum and minimum activity 7 mm was recorded against Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Streptococcus aureus in methanolic extract; Vibrio alginolyticus in EDTA extract. This study reveals that both the methanolic extract of the whole body tissue and EDTA extract of cuttlebone have good antimicrobial activity depending on the concentration.
to cite this article:
S. Vairamani, N. Subhapradha, P. Ramasamy, A. Barwin Vino, S. Raveendran and A. Shanmugam, 2012. Antibacterial Activity of Methanolic Extract of Whole Body Tissue and Ethylene Diamine Tetra Acetate Extract of Cuttlebone of Sepiella inermis (Orbigny, 1848). Research Journal of Microbiology, 7: 263-272.
Received: February 06, 2012;
Accepted: August 01, 2012;
Published: August 31, 2012
The marine organisms are quite interesting in having several new compounds
that show many important biological activities which are under investigation
by researchers all over the world and provide many leads and basic principles
in the development of new pharmaceuticals (Faulkner, 2000a,
b; Da Rocha et al., 2001).
Molluscs are the second largest animal group represented by 100,000 species
so far recorded which includes monoplacophorans to more specialized group, cephalopods
that inhabit almost all the environments and habitats. The class: Cephalopoda
includes the nautilus, cuttlefishes, squids and octopods which are exclusively
in marine and vary in their form, size and nature (Voss, 1973).
Cephalopods are commonly available throughout the world. The cephalopod diversity
in India is represented by 80 species and in Parangipettai 17 species are reported
(Silas et al., 1985; Shanmugam
et al., 2002). These are important as a food resource as well as
in scientific research (Ngoile, 1987). Since there is
an alarming increase in the resistance obtained by the microbes against majority
of the antibiotics under clinical use, it becomes very important to look for
novel natural products showing newer mechanism of action against those microbes
(Bansemir et al., 2006; Ilhan
et al., 2007). Among the invertebrates, the discovered bioactive
compounds in mollusks were identified essentially as peptides, depsipeptides,
sterols, sesquiterpenes, terpenes, polypropionates, nitrogenous compounds, macrolides,
prostaglandins and fatty acid derivatives and alkaloids which presented specific
types of activities (Balcazar et al., 2006).
The crude products extracted from plants and animals are proving themselves
as good source of many drugs or acting as a source of basic active principles
(Kamboj, 1999). Though there are many studies made on
mollusks to report their biomedical importance (particularly antimicrobial),
they are from individual body parts of the mollusks (Rajaganapathi,
2001) like mucus from Achatina fulica (Kubota
et al., 1985); egg mass and purple fluid from Aplysia kurodai
(Yamazaki, 1993); body wall of Dolabella auricularia
(Iijima et al., 2003). In this series of study,
an antibacterial and antifungal peptide was isolated by Charlet
et al. (1996) from the blood of immune-challenged and untreated Mytilus
The mytilin isoforms C, D and G1 were isolated from Mytilus galloprovincialis
and exhibited complementary antimicrobial peptides (Mitta
et al., 2000). In addition, a novel antifungal peptide that delays
the growth of Neurospora crassa and Fusarium culmorum, mytomycin
has been isolated and partially characterized in conjugation with defensins
and mytilins from M. edulis (Charlet et al.,
1996). The increasing resistance of antibiotics by the pathogenic microorganisms
develops the demand for the isolation of novel alternative antimicrobial substances
(Obeidat et al., 2012). Further these compounds
are being extracted not only from the whole animal but also from different body
parts including skeleton (internal shell in the case of cuttlefishes) which
showed many pharmacological properties and hence medicinal value. Keeping the
importance of mollusks as a potential source of many bioactive compounds, the
cuttlefish S. inermis was taken to study the antimicrobial activity of
the methanolic extract of whole body tissue and the polysaccharides extracted
from the cuttlebone.
MATERIALS AND METHODS
Chemicals and reagents: Muller Hinton Agar, Czapek Dox broth, Czapek Dox agar and sterile antimicrobial disc were purchased from Hi-media. Ethylene Diamine Tetra Acetate (EDTA) and Barium hydroxide were obtained from Loba Chem. Company. Acetone and Methanol were obtained from Merck. All other chemicals used were of analytical grade.
Collection of animals: The cuttlefish S. inermis was collected from Thondi landing centre, situated at Lat 9°44N; Long 079°02E, South East coast of India.
Tissue extracts: The extraction process was completed at a few steps. The cuttlebone and ink sac were removed from S. inermis; remaining tissues were cut into small pieces and extracted with 100% methanol for 24-48 h by incubating at room temperature. The extract was centrifuged to collect the supernatant and concentrated under vacuum in desiccators. The crude methanolic extract of whole body tissue was assayed for antibacterial activity using standard disc diffusion method.
Isolation of polysaccharides: The polysaccharide extract was obtained
from the internal shell of S. inermis 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 Hyflo Super Cel. Then saturated Barium hydroxide solution was added to the filtrate and allowed to stand overnight. Then the precipitate was collected on a filter paper (Whatman No.1) with Hyflo Super Cel and washed with distilled water. The dialyzate solution present in the dialysis membrane was then freeze-dried and white colour powder was obtained. The lyophilized powder was used for assaying the antibacterial activity.
Microbial cultures: The ten strains of bacteria used in the present study included Gram-positive: Staphylococcus aureus, Staphylococcus pneumoniae and Streptococcus aureus; Gram-negative: Vibrio cholerae, Vibrio alginolyticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella sp. and E. coli. All the bacterial strains were clinical isolates, obtained from Raja Muthayiah Medical College, Annamalai University.
Extraction and preparation of stock solution: Nutrient broth was prepared and sterilized at 15 lbs pressure for 15 min. Ten bacterial strains were inoculated in the sterilized Nutrient broth and incubated at 37° for 24 h. Mueller Hinton Agar (MHA) were prepared, sterilized in an autoclave at 15 lbs for 15 min pressure and poured into sterile petri dishes to incubate at 37° for 24 h. The 24 h bacterial broth cultures were inoculated in the petri dishes by using a sterile cotton swab. Then tissue extracts were impregnated in the sterile disc (5 mm dia) and after evaporation the discs were placed in respective swabbed plates. Both positive (tetracycline) and negative (methanol) control discs were also placed in all the plates.
The lyophilized powder of crude extract (stock solution) was prepared at a concentration of 1 mg 0.1 mL-1 in 10 mM EDTA. From this 0.24, 0.18, 0.12 and 0.06 mL of sample was taken and each was made up to 0.24 mL with respective solvent to represent the corresponding concentration of 100, 75, 50 and 25%, respectively.
Antibacterial assay: Antibacterial activity of the whole body tissue
extracts and polysaccharides from cuttlebone was determined by the agar disc
diffusion method (Barry, 1980). Briefly, a suspension
of each tested microorganisms was carefully mixed in a tube with 18 mL of MHA
and then poured on petri plates. Sterile filter-paper discs (Whatman No.1; 5
mm in diameter) were impregnated with 50 μL of the extracts. Positive control
discs containing 50 μL of Tetracycline and negative control containing
50 μL of methanol and 10 mM EDTA each were used. These plates were allowed
to dry at room temperature for 3 h and after their impregnation in each petri
plates, the petri plates were incubated at 37° for 48 h. The diameters of
the inhibition zones were measured in millimeters. Each extract was tested thrice
for the confirmation of their activity.
The methanolic extraction of whole body tissue and the polysaccharides from
cuttlebone of S. inermis showed activity against almost all pathogenic
organisms. In general, the activity was higher in 100% concentration and lower
in 25% concentration but activity was absent in negative control and positive
control showed activity against all the bacterial strains (Fig.
||The antibacterial activity of methanolic extract of whole
body tissue and EDTA extract (polysaccharides) from cuttlebone of S.
inermis against (a) Vibrio cholerae, (b) Vibrio parahaemolyticus,
(c) Pseudomonas aeruginosa, (d) Streptococcus aureus, (e)
Klebsiella pneumoniae, (f) Staphylococcus pneumoniae, (g)
Vibrio alginolyticus, (h) Salmonella sp., (i) Staphylococcus
aureus and (j) E. coli
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 methanolic extract of whole body tissue and V. parahaemolyticus alone in EDTA extract from cuttlebone.
In 75% concentration, methanolic extract showed highest activity of 9 mm against K. pneumonia, S. aureus and S. pneumoniae whereas the EDTA extract showed highest activity of 8 mm against V. cholerae, K. pneumoniae and V. alginolyticus. The lowest activity of methanolic extract with 7 mm inhibition zone was observed against P. aeruginosa, Salmonella sp. and E. coli; whereas the EDTA extracts showed 7 mm inhibition zone observed against P. aeruginosa and S. aureus.
In 50% concentration, the maximum activity of 9 mm was recorded against K. pneumoniae and S. aureus in methanolic extract; whereas the maximum activity (11 mm) was recorded against E. coli in EDTA extract. The lowest activity of 7 mm was recorded against P. aeruginosa, Salmonella sp. and E. coli in methanolic extract and 7 mm against V. cholerae, V. alginolyticus and Salmonella sp. in EDTA extracts.
In 25% concentration, only 7 mm of inhibition zone was recorded against: P. aeruginosa, K. pneumoniae, S. aureus and S. aureus in methanolic extract; V. alginolyticus in EDTA extracts. There is no activity in all the concentrations of methanolic extract of S. inermis against V. alginolyticus and EDTA extract from cuttlebone of S. inermis against S. aureus and S. pneumoniae.
In recent years, great attention has been paid to the bioactivity of natural
products because of their potential pharmacological utilization. Most homeopathic
medicines are either of plant or animal origin (Ray and
Mukherjee, 1979). Several molecules extracted from marine invertebrates,
including bivalves, reported broad-spectrum antimicrobial activities, affecting
the growth of bacteria, fungi and yeasts (Mitta et al.,
2000). The worlds oceans, covering more than 70% of the earth surface
represent an enormous resource for the discovery of potential chemotherapeutic
agents. Because of the diversity of marine organisms and habitats, marine natural
products encompass a wide variety of chemical class including terpenes, shikimate,
polyketides, acetogenins and peptides, alkaloids of varying structures and multitude
of compounds of mixed synthesis.
Antibacterial activity has previously been described in a wide range of molluscan
species such as oyster (C. virginica), mussel (M. edulis and Geukensia
demissa), muricid mollusks (Dicathais orbita) and sea hare (Dolabella
auricularia) (Benkendorff et al., 2001).
In most of the species studied, the haemolymph, egg masses or the whole body
have been tested for their activity. However, by far the majority of marine
organisms are yet to be screened and the potential for discovering a useful
antibiotic is sufficient to warrant further research. This study dealt the antibacterial
activity of methanolic extract of whole body tissue and EDTA extract (polysaccharides)
from cuttlebone of S. inermis. The activity was recorded in almost all
the concentration with variation.
The activity of methanolic extract of whole body tissue and EDTA extracts (polysaccharides) from cuttlebone was found to be high in 100% concentration than the three other concentrations. In general, the activity was found to be concentration dependent (Fig. 1).
The methanolic extract of whole body tissue of S. inermis showed activity
against almost all the bacterial strains in the concentrations tested with the
maximum activity of 12 mm in 100% against K. pneumoniae and S. aureus
and the minimum activity of 7 mm in 25% concentration against K. pneumoniae,
S. aureus, P. aeruginosa and S. aureus. The ethanolic extract
of Brillantaisia patula was active against all the tested pathogens such
as S. aureus, Enterococcus faecalis, Proteus hauseri,
P. aeruginosa and E. coli whereas the methanolic extract inhibited
all the bacterial growth except S. aureus (Faparusi
et al., 2012). Hexane extract of Senna didymobotrya showed
16 mm of inhibition zone against Microsporum gypseum (Korir
et al., 2012). Acetic acid extract of Chlorophytum borivilianum
showed maximum antibacterial activity against S. aureus and least
activity against B. subtilis (Sundaram et al.,
The EDTA extract (polysaccharides) from cuttlebone showed antibacterial activity against all the bacterial strains except S. aureus and S. pneumoniae with the activity ranging from 7 mm inhibition zone in 25% concentration against V. alginolyticus to 12 mm in 100% concentration against S. aureus.
The results clearly showed that majority of the extracts exhibited appreciable
antibacterial activity against human pathogens but reported varying activity
against different bacterial strains. The level of activity measured by disc
diffusion assay is dependent on both the rate of diffusion of extract into the
agar and the potency of the extract. Extracts that contain highly active compounds
(more potent) but have physical properties that generate a lower diffusion rate,
may appear to have low activity in the assay (Kelman et
In the crude venom of hypobranchial gland of muricid gastropod Rapana rapiformis,
the highest activity was recorded as 26 mm against K. pneumoniae, 22
mm against V. cholerae and 18 mm against S. aureus (Murugan
et al., 1991). The hypobranchial gland extract of Chicoreus ramosus,
inhibits the growth of ten bacterial strains; out of this, the broad inhibition
zone was formed against S. faecalis and S. aureus (Kagoo
and Ayyakkannu, 1992). The ethanol extracts of hypobranchial gland of C.
virgineus showed 10 mm of inhibition zone against S. typhi, 7 mm
against Shigella flexneri, 6 mm against V. cholerae, 5 mm against
K. pneumonia and 4 mm against B. subtilis and E.
coli; but methanol extract exhibited inhibition only against S. pyogenes
Anand and Edward (2001) recorded 10, 8 and 5 mm inhibition
zone in the case of the ethanol extract of T. delicatula, B. spirata,
T. brunneus and L. arthritica operculum, respectively against
B. subtilis; but the activity was absent against E. coli,
K. pneumoniae, Proteus vulgaris, P. mirabilis, S. typhi,
S. flexneri, S. aureus and V. cholerae. Further the whole
body tissue extracts of Trochus radiatus obtained with different solvents
(acetone, ethyl acetate and dichloromethane) were screened for their antibacterial
activity. All the extracts exhibited clear zones of inhibition for the seven
of the nine human pathogens tested. The highest activity was formed against
Enterobacter aerogenes, S. aureus and E. coli (MaryElizabeth
et al., 2003).
In this context, the similar antibacterial activity was recorded by Rajaganapathi
(2001), who has reported the broad-spectrum antibacterial activity for 13
species of molluscan extracts comprising seven gastropods, one bivalve and five
cephalopods. The activity varied with extracts and bacterial species and the
antibacterial activity was found to be greater in cephalopods than gastropods
and bivalves. In study, the methanol and saline extracts of ink gland, salivary
gland, body mucus and internal shell of cephalopods such as Loligo duvauceli,
Sepia pharaonis, Sepiella inermis, Octopus dollfusi and Cistopus
indicus recorded varying antibacterial activity against the different bacterial
strains viz., B. subtilis, E. coli, K. pneumoniae, P.
vulgaris, P. mirabilis, S. typhi, S. flexneri, S.
faecalis and V. cholerae. All the cephalopod extracts exhibited activity
against at least three bacteria and the highest activity of 10.5 mm was recorded
in the ink gland extract against P. mirabilis. Further, the methanol
extracts of the cuttlebone of S. pharaonis showed activity against
S. flexneri (5 mm), S. faecalis and V. cholerae (4.5 mm) and
S. typhi (3.5 mm); whereas S. inermis extract of cuttlebone showed
activity only against K. pneumoniae and V. cholera (3.5 mm). Such
similar activities were found only in 50, 75 and 100% concentrations of S.
aculeata but the S. brevimana extracts showed highest activity against
all the strains at all concentrations. A wide spectral antibacterial activity
has been recorded in almost all the concentrations of the methanolic extract
than the polysaccharide extract from S. prashadi which explains and supports
the presence of active principle in both the methanolic and polysaccharide extracts
(Ramasamy et al., 2011). Lannea velutina
showed potent bactericidal activity against E. faecalis, B. subtilis,
S. aureus, S. camorum, E. aerogenes, P. mirabilis and
P. aeruginosa (Ouattara et al., 2011).
Aqueous extract of Valeriana wallichii showed maximum activity against
S. aureus whereas the methanolic extract showed highest activity against
B. subtilis (Sati et al., 2011).
The EDTA extract of D. sibogae gladius recorded 10 mm inhibition zone
against E. coli and K. pneumoniae, 9 mm inhibition zone against
S. aureus and 7 mm against S. typhi. Whereas the EDTA extract
of L. duvauceli extract showed only low activity i.e., 5 mm against P.
aeruginosa, 4 mm against S. typhi and E. coli. At the same
time, the gladius extract of both the species showed no activity against V.
cholerae. The EDTA extract from the gladius of D. sibogae recorded
potent antibacterial activity against all the bacterial strain mentioned above
and at the same time the polysaccharide of L. duvauceli gladius extract
recorded only low activity. The EDTA extract from the gladius of L. duvauceli
showed antifungal activity against A. fumigatus, A. flavus and
Rhizopus sp. whereas gladius extract of D. sibogae recorded
the antifungal activity against A. fumigatus and Rhizopus sp.
only. There was no activity for both the species against Candida sp.
(Barwin Vino, 2003). The antibacterial activity was predominant
among cuttlebone extracts (using EDTA) of the cuttlefishes such as S. aculeata
and S. brevimana against almost all the 9 pathogenic bacterial strains
tested viz., B. subtilis and E. coli, K. pneumoniae,
S. aureus, P. aeruginosa and Streptococcus aureus,
V. cholerae, V. parahaemolyticus, S. typhi and Shigella
sp. The activity was recorded in almost all the concentrations except in
control. On comparison the activity was higher in the cuttlebone extract of
S. aculeata than S. brevimana (Shanmugam et
Although different species and experimental procedures were used in the different studies, they indicated the high frequency of detectable antimicrobial activity in marine molluscs. These results enforce the idea that cephalopods are a source to be considered in discovering new substances for drug development. In the present investigation highest inhibition zone of 12 mm was recorded against K. pneumoniae and S. aureus in S. inermis whole body tissue methanolic extract, highest inhibition zone of 12 mm was recorded against Staphylococcus aureus in cuttlebone EDTA extract (polysaccharide). There was no activity seen against V. alginolyticus with methanolic extract whereas no activity was found against S. aureus and S. pneumoniae with cuttlebone EDTA extract. Thus in the present study a wide spectral antibacterial activity has been recorded in almost all concentrations of the extracts which is the significant finding of the study. Further investigations intending to purify these active compound(s) should be considered to clarify their chemical nature.
The methanolic extract of whole body tissue showed activity against 9 human pathogenic bacterial strains except V. alginolyticus. The polysaccharide from cuttlebone showed activity against eight human pathogenic bacterial strains except Streptococcus aureus and S. pneumoniae. The maximum activity of 12 mm in 100% against K. pneumoniae and Staphylococcus aureus in methanolic extract of whole body tissue and the highest activity were recorded 12 mm in 100% concentration against Staphylococcus aureus. Thus, the present study provides the baseline information to the future researchers in this field and also paves the way for the wise utilization of the cuttlebone and cuttlefish meat by the pharmaceutical technologist for the extraction of useful drugs in future.
We are grateful to the Dean and Director of CAS in Marine biology, Faculty of Marine Science, Annamalai University, Parangipettai for given encouragement and support. One of the authors (AS) is also thankful to the Ministry of Earth Sciences, New Delhi for the financial assistance.
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