Post on 23-Jun-2020
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I. GENERAL INTRODUCTION
1.1. Introduction
Oceans cover nearly 71% of earth’s surface and pose nearly three lakh
described species of plants and animals, representing 34-36 phyla and some of them
are exclusively of the marine ecosystem (Pomponi, 1999; Jimeno, 2004; Kijjoa and
Swangwong, 2004). Historical records show that human being has become aware of
the venomous nature of some sea creatures for at least 4000 years (Colwell, 2002).
It has been known for centuries that sponges contain bioactive compounds that are of
potential medical importance. In 19th and 20th centuries, cod liver oil was in use as
supplementary nourishment. However only in middle of 20th
century scientists began
to systematically probe oceans for medicines. By the early 1950s, Ross Nigrelli of the
Osborn Laboratories of the Newyork aquarium (New York Zoological society)
extracted a toxin Holothurin from cavierian organs of the Bahamian sea cucumber,
Actynopyga agassiz which showed some antitumour activity in mice (Nigreli et al.,
1967).
Several marine organisms are sessile and soft bodied with defensive chemical
weapons (Secondary metabolites) for their protection. These compounds help them to
deter predators, keep the competitors at bay and also to paralyze their prey. The
diversity of secondary metabolites produced by marine organisms has been
highlighted in several reviews (Faulkner, 2002; Proksch et al., 2002; Haefner, 2003;
Jimeno et al., 2004; Jha and Zirong, 2004). These compounds belong to different
structural types namely diterpenoids (37%), steroids/sterol glycosides (18%),
sesquiterpenoids (17%) and the remaining were alkaloids, amino acids, fatty alcohol
esters, glycolipids, nucleosides, macrolides, porphyrins, aliphatic cyclic peroxides etc.
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There is ample of evidences documenting the role of these metabolites in chemical
defense against predators (Schupp et al., 1999; Pisut and Pawlik, 2002) and epibionts
(Thakur, 2001; Thakur et al., 2003). Of the immense biological diversity in the sea as
a whole it is increasingly recognized that a huge number of natural products and novel
chemical entities exist in the ocean with biological activities that may be useful in the
quest of finding drugs with greater efficiency and specificity for the treatment of
many human diseases (Mayer and Lehman, 2000; Prosksch et al., 2002). Drugs
derived from unmodified natural products or semi-synthetically obtained from natural
sources corresponding to 78% of the new drugs approved by the (Food and Drug
Administration) FDA between 1983 and 1994 (Gilberto et al., 2001). With marine
species compromising approximately a half of the total global biodiversity, large scale
screening will continue to play a paramount role in the development of new drugs
(Xu et al., 2003).
There are approximately 5000 sponges, 11,000 species of Cnidarians, 9000
species of Annelids, 66,535 species of molluscs and 6000 species of gastropods,
15,000 species of bivalves and 600 species of cephalopods have been reported to
occur in sea (Alfred et al., 1998). This rich diversity of marine organisms assumes a
great opportunity for the discovery of new bioactive substances. Interestingly, these
precious natural products have been obtained from marine microorganisms as well as
invertebrates such as sponges, molluscs, bryozoans, ascidians etc (Thakur and Miller,
2004). The number of potential compounds isolated from marine realm has virtually
soared and this number not exceeds to 10,000 with hundreds of compounds still being
discovered every year (Kisugi, et al., 1992; Wright, 1998; Nuijen, et al., 1999;
Proksch, et al., 2002; Torres, et al., 2002). The marine natural products have been
investigated predominantly for their antimicrobial, cytotoxic, antitumour, antiviral,
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anti leukemic and anti inflammatory properties (Anand and Edward, 2001; Kamiya
et al., 1989; Pettit et al., 1987; Anand et al., 1997; Rajaganapathi et al., 2000). With
the combined effects of marine natural products Chemists and Pharmacologists, a
number of promising identified molecules are already in market, clinical trails or in
pre-clinical trails. A variety of metabolites have reached the clinical tests mainly as
antiviral and antitumour drugs. Marine compounds are also being tested successfully
as insecticides and even for specific pest control. From 1960’s to 1990’s
approximately 300 bioactive marine natural products were filed for patent.
Since the high antiquity, terrestrial plants have been the main if not the only
source of valuable products. Plants and their extracts were used for a variety of
applications. Extensive use and increasing demand have resulted in insufficient plant
material to satisfy market needs and have contributed to destroy some of the natural
bioactive compound are concerned, phytochemistry has almost sources of known
metabolites since popular wisdom had already done this seaving work along the
centuries. On the other hand, synthetic chemical industries were unable to supply
chiral compounds, at low cost to afford new bioactive products completely different
from the “me too” compounds that filled the shelves of pharmaceutical industries.
This situation has deeply contributed to stimulate the increasing interest for
unconventional and thus unexplored sources of natural products.
In this respect marine organisms have received much attention over the past 30
years. Most of the compounds isolated from fish, coelenterates and molluscs were
proteinaceous and /or hydrosoluable. During that period it was very difficult to purify
isolate and identify the compounds due to lack of development of sophisticated
equipments like IR, NMR, Chromatography, GC-MS etc. Tremendous development
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in Science and Technology for the past ten years and the need for new drugs, thirst us
to look into the unique environment, the marine which is a boon of natural chemistry
for the growing medical field. As a result an amazing number of secondary
metabolites has been isolated and described during the past ten years. Most of these
metabolites possess unique skeletons that have no terrestrial counter part and that still
constitute a challenge for natural products chemists.
Dinoflagellates and benthic algae, sponges, coelenterates, molluscs,
echinoderms, tunicates and to a less extend fishes are among the principal sources of
marine bioactive metabolites (Kelecom, 2002). Though the information about marine
natural product chemistry and their ecological and developmental implications are
from the different parts of the world, very little attention has been paid to this aspect
in India. Even though the Indian marine environment is unique for its mega diversity,
to date only a fraction of biodiversity has been properly explored for novel bio-active
compounds that can be developed as new drugs and/or agro chemicals.
For the past 50 years antibiotics have revolutionized life saving medicine by
providing cure for formerly life threatening diseases. However, stains of bacteria and
fungi have recently emerged that are virtually unresponsive to antibiotics. Such multi
drug resistance arising through antibiotic misuse is now recognized as a global health
problem. The situation is exacerbated by the fact no novel chemical classes of
antibiotics have been discovered for twenty years. Although many pre existing
antibiotics have been modified to yield new derivatives, bacteria have the potential to
mutate resistance mechanisms to combat these derivatives (Hancock and Lehrer,
1998).
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In the past 20 years the pharmaceutical industry has been relatively successful
in constraining problems due to single resistance determinants; however the advent of
multiple resistance mechanisms has severely limited the effective use of major class
of drugs (Chopra et al., 1996). The critical events are the emergence of Staphylococcus
aureus with decreased sensitivity to vancomycin, worldwide resistance to penicillin in
Streptococcus pneumonia and multiple resistances to Mycobacterium tuberculosis.
The need for new antibiotics is imperative because resistance to antibiotics in
bacterial population has increased dramatically with time and usage of antimicrobial
drugs. Evidence suggests that development and spread of resistance to any new
antibacterial agent is probably inevitable however, new drug classes with novel
mechanisms of actions will create effective therapy (All Sop, 1998). Commercial
interests in the development of new pharmaceuticals and pharmacological agents have
ensured that this is the most developing area of marine biotechnology. Marine
biotechnology is the science in which marine organisms is used in full or partially to
make or modify products to improve plants or animals or to develop microorganisms
for specific uses. With the help of different molecular and biotechnological
techniques, scientists have been able to elucidate many biological methods applicable
to both aquatic and terrestrial organisms. In spite of the advances in computer assisted
drug design, in molecular biology and gene therapy, there is still a pressing need for
new drugs to counter act drug resistance pathogens.
At this juncture, the molluscs received attention not only for their delicacy for
sea food next to fishes and crustaceans but also as known for the pocession of
bioactive compounds of pharmaceutical interest (Shenoy, 1988). Among the different
molluscs the prosobranchs are proved to be excellent source of bioactive compounds.
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While scrutinizing the literature on this particular group, there is paucity of
information on bioactive compounds with reference to antibacterial and antifungal
properties. This lacuna in the field has leaded the present study with following
objectives.
• To screen the Prosobranchs and select the animals with active bioactive
substances.
• To identify the selected animals upto species level.
• To extract the bioactive substances from the selected (identified) animals.
• To subject the extract by antimicrobial assay on pathogens followed by
purification of extract by Column and Thin layer chromatography,
characterization of most potent probable antimicrobial compounds by FT- IR
( Fourier transform infrared spectroscopy), H¹NMR (Hydrogen Nuclear
magnetic resonance spectrometry), confirmation of compounds by HPLC
(High-performance liquid chromatography) and GC-MS (Gas–
Chromatography- Mass spectrometry) studies and
• Testing the effect of compounds for Pharmacological studies such as
analgesic, antipyretic and anti inflammatory activities.
1.2. Description & characteristics of the collection site
Description of the study area
The Gulf of Mannar (GOM) which extends from Adamsbridge to
Kanyakumari is an established National Marine Park located between India and
Srilanka on the South East Coast of India. The Gulf of Mannar is renowned for being
a biological treasure and storehouse for thousands of species of flora and fauna
largely due to diversified microhabitats for sedentary organisms. The Gulf of Mannar
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with its precious ecosystems such as coral reefs, mangroves, and sea grasses is one of
the highly productive coastal areas in India.
There are about 21 islands covering an area of 625 hectares and the coral reefs
of fringing and patch types extend from Rameswaram to Tuticorin (Lat.8˚50`-9˚10`N
and Long.78˚10`-79˚10`E) covering a distance of 140 km. The islands are located
between the Latitude 8˚47`N - 9˚15`N and Longitude 78˚12`E - 79˚14`E and are
grouped into four namely Mandapam group (7 islands - Shingle, krusadai, Pullivasal,
Poomarichan, Manoliputti, Manoli and Hare), Keezhakkarai group (7 islands - Mulli,
Valai, Thaliyari, Appa, Poovarasanpatti, Valimunai, and Anaipar), Vembar group
(3 islands - Nallathanni, Puluvinichalli and Upputhanni) and Tuticorin group
(4 islands - Kariyachalli, Vilanga Challi, Koswari, and Vaan). The GOM is rich in its
biological resources such as 147 species of Seaweeds (Kaliaperumal, 1998),
13 species of seagrasses (Rajeshwari and Anand 1998), 17 species of Sea cucumbers
(James, 2001), 510 species of finfishes (Durai Raj 1998), 106 species of shell fishes
such as crabs (Jayabaskaran and Ajmalkhan, 1998), 4 species of shrimps (Ramaiyan
et al., 1996) and 4 species of lobsters (Susheela 1993). During the survey of Molluscs
by Deepak and Patterson (2004) recorded 5 species of Polyplacophorans, 174 species
of bivalves, 271 species of gastropods, 5 species of Scaphopods and 16 species of
Cephalopods. Tuticorin (8º45´N; 78º46´E) belongs to Gulf of Mannar area in
Tamilnadu, along the south east coast of India. It is an important seaport in South
India and has its reputation as Pearl City because of pearl fishing. The sea bottom is
rocky in patches, and is generally composed of sandy mud.
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Characteristics of the study area
Introduction
The total life of the world depends on water and hence the hydrological study
is very much essential to understand the relationship between its different trophic
levels and foodwebs (Soundarapandian et al., 2009). Environmental conditions such
as topography, water movement and stratification, salinity, oxygen, temperature and
nutrients of particular water mass also determine the composition of its biota
(Karande, 1991). Oceans are very complex environment with great variations in
temperature, salinity, pH, dissolved oxygen and nutrients. The physico-chemical
parameters play a major role in the ecosystems to maintain the biodiversity profile of
the marine environment. Hence the present study has been designed to investigate the
seasonal changes of physico-chemical parameters at the collection area of the
screened animals.
MATERIAL AND METHODS
The present investigation was carried out at monthly intervals for one year
from April 2010 to March 2011. The water samples were collected in clean glass
containers and brought to the laboratory. For the estimation of dissolved oxygen the
samples were fixed on the spot and then estimated following Winkler’s method as
described by Strickland and Parsons (1972). Temperature was recorded with the help
of Celsius thermometer. The salinity was estimated using a Refractometer (ATAGU,
Japan). The hydrogen-ion-concentration (pH) of the water was measured by pH Pen
(HAMNA, Italy). Data on monthly rainfall at the study area was obtained from the
Indian Meteorological Department at Tuticorin.
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RESULT
Rain fall
Monthly variations in the rainfall during the study period (April 2010 - March
2011) are presented in the Figure (1). The maximum rainfall of 300.9mm was
observed during November (monsoon) 2010 and the minimum of 4.4 mm in June
(summer) 2010. No rainfall was recorded during February and March 2011.
Temperature
Monthly variations in atmospheric temperature are depicted in Fig.2.
Atmospheric temperature fluctuated between 30˚C and 37˚C. The highest temperature
(37˚C) was observed during summer season (April, May 2010) and the lowest
temperature (30˚C) in monsoon season (November and December 2010).
Water temperature
The water temperature varied from 27˚C to 32˚C (Fig.3). Maximum surface
water temperature (32˚C) was noted during summer season (April, May and
June 2010) and minimum was recorded during monsoon season (November 2010).
Dissolved oxygen
Monthly variations in oxygen values are shown in Fig.4. The highest dissolved
oxygen content of 6.398 ml/l was recorded during monsoon season (November 2010)
and the lowest value (3.7477 ml/l) was recorded during summer season (May 2010).
pH
Water pH values were ranging from 6.7 and 8.3 (Fig 5) and the highest (8.3)
pH value was observed during April, May and June 2010 (summer) and the lowest
value was recorded during November and December 2010 (6.8) (monsoon).
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Salinity
In the present study the salinity values of the water ranged between 31 and
36.5 (Fig 6). Maximum salinity was recorded during summer (April and May 2010)
and minimum salinity was recorded during monsoon (October and November 2010).
DISCUSSION
The environmental conditions such as temperature, oxygen, pH, salinity and
also nutrients influence not only the composition of its biota but also the growth and
distribution of species of flora and fauna (Karanade, 1991 and Swami et al., 2000).
Extrinsic and intrinsic factors are known to influence the distribution and abundance
of the screened Prosobranchs in the Gulf of Mannar.
North east monsoon during November 2010 was responsible for rainfall
at Tuticorin coastal region in Gulf of Mannar. Seasonal changes in rainfall influence
the density of lower invertebrate population of the intertidal areas (Odum 3rd
Ed.,
1971). Atmospheric and water temperature fluctuated seasonally. The low
temperature could be due to strong land breeze, rainfall, land run-off, and cloudy sky
as reported by Ananthan (1994). The temperature variation is one of the factors in the
coastal estuarine system, which may influence the physico-chemical characteristics
and also influence the distribution and abundance of flora and fauna
(Soundarapandian et al., 2009).
Dissolved oxygen content showed seasonal fluctuation, and it varies due to
photosynthesis and respiration by organisms. Percent saturation of oxygen depends
strongly on temperature as cold water holds more oxygen. The highest level of
dissolved oxygen observed during monsoon might be due to phytoplankton
production and photosynthesis as reported by Santhosh Kumar and Perumal (2011).
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An earlier report from these areas coincides with the present study, revealing the high
contents of dissolved oxygen during monsoon (Thilaga, 2005). Low dissolved oxygen
observed during the summer season could be attributed to the lesser input of
freshwater into the study area as reported by Thilaga (2005).
pH showed only slight variation during the present observation. The highest pH
values during summer could be due to uptake of CO2 by photosynthesizing organisms
(Balasubramanian and Kannan, 2005). Generally pH is quite lower in rainy season as
compared to winter and summer may be due to the influence of freshwater influx,
dilution of sea water, low temperature and organic matter decomposition as suggested
by Santhoshkumar and Perumal (2011). Similar trend in pH was reported by
Seenivasan (1998) from the Vellar estuarine system, and Ananthan (1994) from
Pondicherry coastal water. Rajasegar (2003) and Mohamed Meeran et al., (2011) also
stated that pH profile altered with seasons and the pH of water also gets drastic
change with time due to exposure to biological activity and temperature..
Salinity in the study area was higher during summer which could be described
to the higher degree of evaporation and less tidal action. The present findings are in
affirmative with that of Mohammed Meeran et al., (2011). Similar trend in the salinity
values were also observed from various parts of south east coast of India (Mitra,
1990; Palanichamy and Rajendren, 2000; Sulochana and Muniyandi, 2005;
Sundaramanickam et al., 2008; Soundarapandian et al., 2009). The monsoon driven
current systems govern the seasonal distribution of salinity in these waters. The lower
salinity during monsoon could be due to inflow of fresh water through rainfall as
reported by Sulochana and Muniyandi, 2005. Salinity is known to play a key role in
the distribution of marine organism in near shore and estuarine region, where
fluctuations in salinity are well pronounced (Ajmal khan and Natarajan 1981).
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The existing physico-chemical characteristics from the study area were found
to be conducive for the survival of prosobranch molluscs.
1.3. Screening of molluscs from Gulf of Mannar
Some commonly available Prosobranch gastropods like Tonna galea, Ficus
gracilis, Bursa rana, Murex ternispina, Chicoreus virgineus, Purpura persica, Thais
rudolphi, Rapana rapiformis, Babylonia spirata, Fusinus longicaudatus, Babylonia
zeylanica and Turbo bruneus were collected initially for the present study. From these
animals basic antimicrobial screening assays were carried out. After scrutinizing the
results such as Babylonia zeylanica, Purpura persica and Chicoreus virgineus
showing maximum antimicrobial activity alone were selected for further study.
1.4. Systematic position
Babylonia zeylanica (Bruguiere, 1789)
Phylum : Mollusca
Class : Gastropoda
Order : Neogastropoda
Super family : Muricoidea
Family : Babylonidae
Genus : Babylonia
Species : zeylanica
Common name: Indian Babylon
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Plate 1 Shows the dorsal and ventral views of the shell of Babylonia zeylanica
The shell is fusiform, slender, thin, and smooth without ribs or sculpture.
It has a large body whorl and a high spire with fine axial lines. The sutures are well
marked, a large lanceolate aperture with a short siphonal canal. The pattern is a
distinctive irregular arrangement of large and small with regular blotches and flames,
decorated with spiral rows of brown to light brown blotches and flames on a pale
ground. Surface is smooth and glossy. Colour is creamy white. Spires are not grooved
and apical spire is purple in colour. Columella growth is usually absent but seldom as
a small ridge near the anterior part of columella ridge. Umbilical opening is not
callused but open with one or two rows of blunt spiny growth along the outer margin.
Anterior part of aperture and umbilicus is light violet. The suture is not sunk and in
which the spire is more strongly elevated and the umbilicus toothed on its outer side.
Foot is large, without posterior appendages. Apex of aperture is constructed by spiral
ridge on inner side only. This edible B.zeylanica is found at muddy bottoms of deeper
waters beyond 50m and mostly found at the east coast of India.
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Systematic position
Purpura persica (Linnaeus, 1758)
Phylum : Mollusca
Class : Gastropoda
Order : Neogastropoda
Superfamily : Muricoidea
Family : Thaisiidae or Muricidae
Sub family : Rapaninae
Genus : Purpura
Species : persica
Common name : Persian perpura /Smooth thaid
Plate 2 Shows the dorsal and ventral views of the shell of Purpura persica
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Shell is spindle in shape with a broad middle region. Shell surface is more or
less even without nodules and about four to five flat but feebly raised spiral ridges on
the body whorl. Of these spiral ridges, two of the uppermost are distinct. The spaces
between the ridges are uniform and finely grooved. The spiral ridges are alternatively
banded white and dark brown. Aperture is comparatively large and the outer lip is
slightly but sharply bent near the posterior end. Outer lip is thin with the inner surface
finely grooved in line with the outer fine spiral ridges. Columella and parietal wall are
heavily calloused and yellowish. The interior of the aperture is white and the inner
edge of the outer lip is banded dark brown with yellow streaks in between, a distinct
character that could be used thaid species of the east coast. It is usually found in tide
pools between rocky reefs and other sheltered regions.
Systematic position
Chicoreus virgineus (Roding, 1798)
Phylum : Mollusca
Class : Gastropoda
Order : Neogastropoda
Superfamily : Muricoidea
Family : Muricidae
Genus : Chicoreus
Species : virgineus
Common name : Virgin murex
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Plate 3 Shows the dorsal and ventral views of the shell of Chicoreus virgineus
The shell is almost triangular, elongate, thin, and strong. Body whorl is board.
It is more angular and sculptured with spines and shorter fronds (the spiny
outgrowths) on the three varices and these outgrowths are closed. Similar spiny
outgrowth is also prominent along the outer lip. The absence of spiny outer lip
differentiates variety ponderosa from C.virgineus. Color is usually white or creamy
with occasional brown bands. The columella is pink and lip has faint denticles. There
is one prominent axial node between varices. C.virgineus inhabits intertidal or
shallow sub tidal zone of muddy sandy bottom.
1.5. Review of literature
The ocean is considered to be a source of potential drugs. The first attempt to
locate the antimicrobial activity in marine organisms was initiated around 1950’s
(Berkholder and Burkholder, 1958) since this time a large number of marine
organisms from a wide range of phyla have been screened for antimicrobial activity.
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Toxic fishes, molluscs, Jelly-fish induced dermatitis, ichtyotoxic red tides,
antihelminthic algae were known in some cases for thousands of years, it was not
before 1960 that a scientific meeting discussed the potential value of the seas for food
and drugs supplies (Nigrelli, editor 1960). Marine organisms exhibit a wide range of
biological activities (Xiang et al., 2004; Wilsanand et al., 2001). Antibiotic (Grein
and Meyer 1958), antiviral (Gustafson et al., 2004), antifungal, antimicrobial
activities of marine organisms have been reported by many authors (Selitrennikoff,
2001; Pan et al., 2004; Tincur, 2004; Todd Narshy, 2006). Compared to the works
carried out in other invertebrate phyla, especially other classes of mollusc, the existing
literature on prosobranch mollusc is meagre.
Several compounds extracted from marine invertebrates posses broad
spectrum antimicrobial activity affecting the growth of bacteria, fungi, and yeasts
(Nakammara et al., 1998; Zasloff, 2002; Thilaga, 2005). Some of the molecules
responsible for these compounds are peptides, terpenes, polypropionates, nitrogenous
compounds, polypeptides, macrolides, prostaglandins and fattyacid derivatives,
sterols and miscellaneous compounds (Maktoob and Ronald, 1997). Charlet et al.,
(1996) worked on mollusc Mytilus edulis (mussel) and purified antibacterial and
antifungal peptides, defensins A and B which shows a high degree of similarity with
arthropod defensins, a large family of cysteine – rich cationic peptites. From the sea
hare Dolabella auricularia, an extremely potent anticancer compound was discovered
(Pettit et al., 1987).
The highly bacteriostatic compound tyriverdin (Prota, 1980) and the mildly
anti microbial oxidation product G-bromoisatin (Fouquet and Bielig, 1971) were
isolated from the egg masses of several muricids. Bioactive metabolites from
molluscs such as sea hare (Schmitz, et al., 1993), Chromodoris sp., (Morris et al.,
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1990), Onhidella sp. (Ireland et al., 1993), were isolated and structurally elucidated.
Macrolides isolated from Hexabranchus sanguineus nudibranch ( Pawlik, 1992);
Inorulides-A-C isolated from Chromodoris inornata nudibranch (Miyamoto et al.,
1999); Aplysianin-E isolated from Aplysia kurodai (Lijima et al.,1995); Kelletinin-A
isolated from Buccinulum corneum (Silvestri et al.,1995);Labdane diterpene isolated
from Trimusculus perurianus gastropod (San-Martin et al.,1996);Oxalic acid and
Oxytetracycline isolated from Mytilus edulis (Shell blue mussel) (Pouliquen et al.,
1997); Hydrolyzate obtained from Perna indica (Brown mussel) (Chatterji et al.,
1999); Antimicrobial glycoprotein (Ogawa et al., 1999); Dactylomucin-P isolated
from Aplysia dactylomela (sea hare) (Melo et al. , 2000); Achacin obtained from
Achatina fulica giant African snail ( Hyun-Sun Kim, 2007).
From Leminda millecra a nudibranch, thirteen compounds including
sesquiterpenes (Kerry et al., 2001) millecrone (Beukes 1998, et al), cubeberone
(Hooper et al 1995) and algoafuran (Gray et al., 2000) were isolated. The
hypobranchial glands of Chicoreus virgineus and egg capsules of Rapana rapiformis
extracted with polar solvents like ethanol and methanol also have been reported to
show wide spectral antibacterial activities (Anand et al., (1997). The presence of
antimicrobial peptide activity in mollusca has been reported from the mucus of the
giant snail Achantina fulica from the egg mass and purple fluid of sea hare Aplysia
kurodai (Lguchi et al., 1982; Kubota et al., 1985). The egg capsules of all muricids
were found to contain tyrindoleninon which is the major antimicrobial metabolite
isolated from fresh egg mass of Dicathais orbita (Benkendorff et al., (2000).
The first marine product to enter clinical trial as an anti tumor agent
Didemnin B, a cyclic antiproliferative depsipeptide from the Caribbean tunicate
Trididemnum soliderm was isolated by Hochster et al., (1998). Many studies on
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bioactive compounds from molluscs exhibiting antitumor, antileukemic and antiviral
activities have been reported world wide (Bai et al., 1990; Madden et al., 2000;Kagoo
and Ayyakannu 1992; Rajaganapati, et al., 2000). Antibacterial activity was reported
against Staphylococcus aureus and Esherichia coli on Trochus radiatus by Mary
Elizabeth et al., (2003). Lamellarins are a group of polycyclic pyrole alkaloids that
were isolated from marine molluscs. Since the first isolation in by Faulkner (1995)
more than 36 kinds of structurally relative lamellarins have been reported (Krishnaiah
et al., 2004 and Ruchirawat et al 2001), which exhibited a wide array of interesting
and significant biological activities such as cell differentiation, inhibition,
cytotoxicity, HIV-1, integrace inhibition and immuno modulation. Lamellarin H. one
member of Lamellarina family has been shown to be active against the topoisomerase
of the MCV and to have anti-HIV properties (Bailly, 2004). Chellaram and Edward,
(2009) isolated a bioactive compound from gastropod Drupa margariticola.
Activities of ethanol extracts of gastropods Babylonia spirata and Turbo
brunneus observed maximum activity against Esherichia coli, Klebsiella pneumoniae,
Proteus vulgaris and Salmonella typhi (Prem Anad et al., 1997). The Mytilin isofarms
C, D and G were isolated from Mytillus galloprovincialis exhibited complementary
antimicrobial properties (Mitta et al., 2000). Dolastatins – a group of cyclic and linear
peptide isolated from marine mollusc Dolabella auricularia (Pettit et al., 1981) which
is an active anticancer natural substance at that time with an ED50 of 4.6 x 10.5 mg/ml
against P388 cell line. Dolastatin 10 inhibits tubulin polymerization and tubulin
dependent GTP hydrolysis (Bai et al., 1990; Madden et al., 2000). Depsipeptide
dolastatin 11 arrests cells of Cytokinesis by causing a rapid and massive
rearrangement of the cellular actin filament network (Bai et al., 2001). Pronounced
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antimicrobial activity was reported by Jeyaseeli et al., (2001) in four bivalves against
Bacillus subtilis.
Highest antibacterial activity was observed against Klebsiella pneumoniae and
Staphylococcus epidermis by the column purified whole body acetone extract of
winged Oyster Pteria chinensis and against Salmonella paraptyphi by the extract of
chloroform of the same animal (Chellaram et al., 2004). Cypraea errones showed
moderate antibacterial and antifungal activity against Staphylococcus aureus,
Streptococcus pyogenes, Aspergillus niger and Candida albicans and very good
activity against Shigalla flexneri in heptane fraction (Prem Anand and Patterson
Edward, (2002).
Ulrich et al., (2001) showed the presence of heparin in the labial palp, ctenidia,
siphons, pallium, intestine and foot of northern quahog clam Mercenaria mercenaria
by histological localization. A compound as powerful as penicillin extracted from
eggs of sea snail (dog whelk) was found to be active against Staphylococcus sp.,
Escherichia coli, Candida albicans and Klebsiella pneumonia. Hypobranchial gland
of Chicoreus virgineus had shown broad spectrum anti bacterial activity
(Rajaganapathi 2000). The methanol extract from the whole body of Hemifusis
pugilinus exhibited activity against Bacillus subtilis, Esherichia coli and Klebsiella
pneumoniae (Rajaganapathi 2000). Methanol and Ethanol 10:10 fraction of Tibia
delicatula showed significant antimicrobial activity on human pathogens (Anand and
Edward, 2001). The lectin from the heart muscle Modiolus modiolus exhibited a
strong antibacterial activity against Vibrio strains (Tunkijjanukij and Olafsen, 1998)
and the gigalins (H and E) from the Oyster Crassatrea gigon acted as opsonins to
stimulate invitro phagocytosis of the marine bacteria Vibrio anguillarum
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(Olafzen et al., 1998). The lectin from Crassostrea virginica agglutinated a large
variety of bacterial species (Fisher and Dinuzzo, 1991).
Annamalai et al., (2007) studied the antibacterial activities of water, ethanol,
methanol, acetone, hexane and butane extracts on Perna viridis and Crassostrea
madrasensis and observed the highest activity in Esherichia coli, Staphylococcus
aureus and Proteus mirabilis. Several molecules extracted from marine invertebrates
including bivalves possess broad spectrum antimicrobial activities affecting the
growth of bacteria, fungi, and yeasts (Mitta, et al., 2000; Nakamura, et al., 1998;
Zasloff, 2002). Antimicrobial activity was detected in various tissues of the horse
mussel Modiolus modiolus (Hang et al., 2004). Antimicrobial activity has been
described in a wide range of species Crassostrea virginica and C. gigas (Anderson
and Beaven, 2001; Mitta, et al., 2000) and a number of bioactive compounds isolated
from the marine gastropod Telescopium telescopium acted as a biomarker to study the
nature of sperm plasma membrane (SPM) as well as its heterogeneity that undergo
changes during their maturation phases (Manik Lal HEMBRAM et al., 2009). The
acetone extract of Siphonaria lessoni was subjected to open column chromatography
on silica gel, using increasing proportions of ethyl acetate in petrolium ether to afford
a new polypropionate the mixtures of the known compounds. Antibacterial and
antiviral activities have been described in the haemolymph of several molluscan
species including several sea hares, sea slug, oysters, and mussel’s species (Gueguen
et al., 2006; Maktoob and Ronald, 1997; Olicard et al., 2005; Roch et al., 2008).
Mytilin B, a synthetic antibacterial peptide from the mussel Mytilus galloprovincialis
exhibited both antibacterial and antiviral activities (Roch et al., 2008).
Ojika et al., (1990) isolated Aplydilactone a novel fatty acid metabolite from
the marine mollusc Aplysia kurodai. Mytimicin a novel antifungal Cys-rich
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polypeptide of 6.2 kDa that hindered the growth of the fungi was isolated and
partially characterized from Mytilus edulis (Charlet et al., 1996). Dolastatin 10,
a highly unusual polypeptide comprising of new amino acids, (2R, 3R, 4S)
-dolaproline, (2R, 4S, 5S) -dolaisoleucine, L-dolapherine and L-dolavaline were
shown to be highly antifungal against Cryptococcus neoformans with MIC 0.37
µg/ml, but not against other fungi (Pettit et al., 1998). Kahalalide F an unusal cyclic
depsipeptide containing (Z)-2-amino-2-dehydrobutric acid and L- orinithine
accumulated by the Hawaiian sacoglassan Elysia rufescens from the green algae
Bryosis pennata is highly cytotoxic as well as strongly anti fungal against Candida
albicans, Cryptococcus neoformans and Aspergillus fumigatus (Shilabin et al., 2007)
An antifungal compound Aplysianin E was isolated from egg mass of sea hare
Aplysia kurodai by Lijima et al., (1995). Aplyparvunin A, a bioactive acetogenin was
isolated from the sea hare Aplysia parvula by Miyamoto et al., (1995). Dolabellin B2,
a 33-residue AMP isolated from the sea hare Dolabella auricularia is fungicidal
against Saccharomyces cerevisiae while it is fungistatic against Candida albicans
(Lijima et al., 2003). Polysaccharids extracted from the cuttlebone of Squatina
aculeata and Sepia brevimana showed promising antibacterial and anti fungal activity
against the human pathogenic strains (Shanmugan et al., 2008). A big defensin named
AiBD of the scallop Argopectan irradians has been cloned and expressed, the
recombinant AiBD (120 residues) was reportedly not only high anti bacterial but also
fungicidal, though detailed fungicidial was not available (Zhao et al., 2007). Many
antimicrobial screening compounds have shown the Gram negative bacteria are more
sensitive than gram positive bacteria (Tadesse et al., 2008).
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The IR spectrum Chilean mollusc Siphonaria lessoni acetone extract of
compounds presented bands at 3460 and 1700 cm-1 indicating the presence of a
hydroxyl and carbonyl groups. The 13 C - NMR spectral data indicated 19 carbons
and, together with an accurate mass spectra measurement (lreims: 296.2571),
indicated the molecular formula C19H36O2 with two sites of instauration. The 13 C
NMR decoupled spectrum showed well resolved resonances for all 19 carbons (Juana
Roviro, et al., 2006). Indole derivative of 6, 6’ dibromoindigo found in different
muricids and it has been demonstrated that these derivatives can have anti microbial
activity (Benkendorff et al., 2000 and 2001a). It has been reported that
6-bromoindiration is a strong GSK inhibitor (Meijer et al., 2003). Several
investigations on the hypobranchial glands of the muricid family have revealed that
the number and nature of precursors involved in the production of tyrian purple
differs among species (Benkendorff et al., 2001a).
GC/MS analysis of the milk from Plicopurpura panda revealed four
compounds that could be identified as tyrindoleninore 3, 6-bromoisatin, 6-bromo-
2-methylsulfinyl-3H indole-3-one and 6-bromo-2-methoxy-3H-indole-3-one (Felipe
Javier, et al., 2009). From the hypobranchial glands of Plicopurpura columallaris
6-bromo-indaline-2-one was detected by Lopez Chavez et al., (2009). Fusariyrous
A and B and haminols a novel polypropionates were isolated from the Mediterranean
molluscs Haminoea fusari by Adele Cutignano et al., (2007). A calcium independent
lectin of molecular mass 47 kda was purified from the foot muscle of marine bivalve
Macoma birmanica (Mausumi Adhya, 2009). Polypropionates are the common
secondary metabolites of the sub classes Pulmonata and Opisthobranchia (Darias
et al., 2006). Two novel triterpenoids aplysoils A and B have been isolated together
with structurally related known metabolites from a South China collection of the
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anaspidean molluscs Alysia dactylomela (Emiliano Manzo et al., 2007). The
Scaphander ligarius collected along the Southern coasts of Italy gave variable
mixtures of dihydrolignarenone and dihydrolignarenone (Giorgio Della Sala et al.,
2007).
In molluscs haemocytes are predominantly responsible for innate immune
defense and release AMPs (Bulet et al., 2004; Tincur and Taylor 2004). AMPs have
been reported from some bivalves and Opisthobranchs gastropods. Defensins were
identified in haemocytes of the mussel Mytilus galloprovincialis (MGD 1and 2)
(Mitta et al., 1999) and in the mantle tissue of the oyster Crassostrea gigas (Cg- Def)
(Gueguen et al., 2006; Gueguen, et al., 2007) respectively. MGDs and Cg-Def
inhibited the growth of the fungus Fusarium oxysporum. Polyporine type AMP
(47 residues) isolated from the Chilean scallop Argopectan purpuratus showed
antifungal activities against Fusarium oxysporum and Saprolegnia parasitica (Arenas,
et al., 2009).
Peptides Achatin I and Achatin II involved in the control of muscle
contraction have been isolated from the African Snail Achatina fulica (Fujimoto et al.,
1991). Fulicin, a peptide isolated from the ganglia of this snail was found to be a
potent stimulator of the contraction of the penis retractor muscle (Ohta, et al., 1991).
A novel polypropionate onchidione was isolated from a marine pulmonate molluscs
Onchidium verruculatum (Marianna Carbone et al., 2008). EPA (Eicosapentaenox
acid) and lectins from marine species have also been used to reduce cholesterol,
triglyceride, and low-density lipoprotein levels in blood and to keep the blood
circulatory system normal. With anti-aggregatory properties, EPA helps in lowering
blood viscosity and is used as a potent antithrombic agent. Some species examined
from Indian coastal regions are rich sources of EPA and DHA (docosaexaenoic acid)
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and are being used in better recovery of cardiac problems. Potent medicines, prepared
from such species as the air-breathing fish mudskipper (Bolephthalmus boddeaerti), a
bivalve (Macoma birmanica), and a gastropod mollusc (Telescopium telescopium)
have been marketed globally (Manik Lal HEMBRAM et al., 2009).
Today, most infectious diseases can be brought under control with natural or
synthetic products. We are still in great need of safer, cheaper and effective drugs.
Some marine molluscs have shown more pronounced activities, useful in the
biomedical area. The potential of marine molluscs as a source of biologically active
products is largely unexplored in India. From the above literature it is understood that
the potential of prosobranchs as a source of biologically active products is largely
unexplored. Hence broad based screening of prosobranchs (gastropods) for bioactive
compound is necessary. Considering the importance of the group and paucity of
information in this line, present study has been undertaken to ascertain the
antibacterial and antifungal activities of body tissue extract of Balylonia zeylanica,
Purpura persica and Chicoreus virgineus and the substance which is responsible for
the antibacterial, antifungal, analgesic, antipyretic and anti-inflammatory activities
was isolated, purified and characterized.
1.6. Work Plan
Available literature reveals that there is paucity of information on the selected
gastropods from the Gulf of Mannar region, but the research work so far carried out
focussed on Ophisthobranchs and Nudibranchs molluscs. The bioactive compounds
are thought to be concentrated more in the predators and coral associated molluscs
(the soft bodied and sessile marine organisms) that have defensive chemical weapons
(secondary metabolites) for their predation and protection. There is ample evidence
documenting the role of these metabolites in chemical defense against predators
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(Pisut and Pawlik, 2002; Thakur, 2001; Thakur et al., 2003). Of the immense
biological diversity in the sea as a whole, it is increasingly recognized that a huge
number of natural products and novel chemical entities exist that may be useful in the
quest of finding drugs with greater efficacy and specificity for the treatment of many
human diseases (Mayer, 2005 and 2007; Proksch et al., 2002). With this back ground,
the present study was planned to understand the bioactive potentials of the selected
gastropods of Gulf of Mannar coastal waters and the results have been presented in
ensuing chapters.
First Chapter deals with general introduction, description and characteristics of
the collection sites, screening of molluscs, systematic position, description of the
screened molluscs and reviews of literature.
The second chapter deals with preparation of crude extracts of whole selected
animals, purification of crude extracts by CC and TLC, testing the extract against
bacterial pathogens and selection of active extract.
Third chapter covers with testing of crude and column purified extracts of
selected animals against fungal pathogens and selection of most potent extract.
Fourth chapter includes the characterization of antibacterial and antifungal
compounds by IR, NMR, H¹NMR, chemical characterization and structural
elucidation of the active purified compounds by GC-MS and confirmation of some of
the probable antimicrobial compounds by HPLC.
Fifth chapter pinpoints with the pharmacological characters of the identified
compounds by analgesic, anti pyretic and anti inflammatory activities of the silica gel
column purified extracts.