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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.