6. . BIOASSAY GUIDED FRACTIONATION AND ... ... 96 6. . BIOASSAY GUIDED FRACTIONATION AND STRUCTURAL

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    INTRODUCTION

    Marine organisms have formed a valuable source of new bioactive

    compounds, many of which are being used in the treatment of many diseases

    and serve as compounds of interest both in their natural form and as a

    template for synthetic modification (Supriya and Yogesh, 2010). The biological

    and chemical diversity of the marine environment represents unlimited

    resource of new active substances in the field of the development of bioactive

    natural products (Aneiros and Garateix, 2004). Antimicrobial peptides are

    generally effective components for developing innate immunity and they are

    ubiquitous in both plant and animal kingdom (Boman, 2003; Ganz, 2004).

    The competitive marine environment enables the organisms to produce

    potent compounds of ecological relevance (Aneiros and Garateix, 2004). The

    marine compounds possess varied functions of interest to humans like

    anthelminthic, anti-bacterial, anticoagulant, anti-diabetic, anti-fungal, anti-

    inflammatory, anti-malarial, anti-platelet, anti-protozoal, anti-tuberculosis and

    anti-viral (Mayer and Hamann, 2005). More than 20,000 natural products have

    been discovered from marine organisms since 1960s (Gu-Ping et al., 2011).

    The main source of bioactive metabolites are invertebrates like

    sponges, jelly fish, sea anemones, corals, bryozoans, molluscs, echinoderms,

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    tunicates and crustaceans (Bhakuni and Rawat, 2005). The marine secondary

    metabolites possess unique chemical novelty and could be categorised into

    steroids, terpenoids, isoprenoids, nonisoprenoids, quinones, brominated

    compounds, nitrogen heterocyclics and nitrogen sulphur heterocyclics. Based

    on the chemical structure of the compounds isolated during 1985 to 2008, Gu-

    Ping et al. (2011) reported that these natural products could be divided into

    seven classes: terpenoids, steroids (including steroidal saponins), alkaloids,

    ethers (including ketals), phenols (including quinones), strigolactones, and

    peptides. Terpenes are the dominant among the isolated marine compounds

    followed by alkaloids (Gu-Ping et al., 2011). Spongouridin and

    spongothymidin are the first marine compounds isolated from the sponge

    Cryptotethya crypta (Bergmann and Feeney, 1951) followed by

    prostaglandins from the Caribbean gorgonian Plexaura homomalla

    (Weinheimer and Spraggins, 1969).

    Bryozoans are a rich and excellent source of novel and biologically

    active secondary metabolites (Faulkner, 2001). Though over 8000 species

    were known from the Phylum Ectoprocta (Bryozoa), the number of natural

    compounds isolated from this group compared to other invertebrates is

    limited. It represented only 1% of natural products reported (Blunt et al.,

    2003). The most well known bryozoan compounds are the bryostatins (Pettit,

    1991), flustramines (Carle and Christophersen, 1980), securamines and

    securines (Rahbaek et al., 1996), tambjamines (Carte and Faulkner, 1983),

    amathamides (Blackman and Matthews, 1985), amathaspiramides (Morris

    and Prinsep, 1999), convolutamydines (Kamano et al., 1995) and

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    alternatamides (Lee et al., 1997). More than 15 tryptophan-derived alkaloids

    have been isolated from Flustra (Holst et al., 1994a). The brominated pyrrolo

    indole deformylflustrabromine was isolated from the bryozoan Flustra foliacea

    from North Sea (Nicola et al., 2002). New cytotoxic oxygenated sterols were

    isolated from the marine bryozoan Cryptosula pallasiana (Tian et al., 2011).

    In the present study, the active metabolite from the crude diethyl ether

    extract of bryozoan Zoobotryon verticillatum was isolated through bioassay

    guided fractionation through chromatographic techniques and an attempt was

    made to elucidate the structure of the active compound with the help of

    Fourier Transform Infrared spectrometry (FTIR), Carbon and Proton Nuclear

    Magnetic resonance (NMR) and Mass spectrometry (MS).

    MATERIALS AND METHODS

    The collection and extraction of bryozoan Zoobotryon verticillatum with

    diethyl ether was carried out as described in Chapter 4. The human bacterial

    pathogenic strains and the antibacterial assay detailed in Chapter 4 were

    used for bioassay guided fractionation.

    Partitioning

    The crude diethyl ether extract was partitioned to assess the polarity

    and to localize the active component (Riguera, 1997; Wright, 1998). The

    concentrated crude extract was partitioned between ethyl acetate and water

    and then, this water phase was subsequently partitioned against n-butanol

    (Wright, 1998; Slattery et al., 1995). Then, the 3 phases (Ethyl acetate,

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    butanol and water) were collected separately; evaporated, concentrated and

    antibacterial assay was carried out against 10 human bacterial pathogens in

    triplicates using 100 µg/disc concentrations.

    Thin Layer Chromatography (TLC)

    The crude diethyl ether extract was also eluted using Thin Layer

    Chromatography plates (pre-coated silica gel plates E–Merck, Germany, Art

    5554 Kiesel 60F254 with 0.2 mm thickness) (Gibbons and Alexander, 1998)

    with different solvent combinations to evolve solvent elution scheme for

    column fractionation.

    Column fractionation

    The active crude diethyl ether extract was partially purified using

    column chromatography by following the method of Wright (1998). The crude

    extract was fractionated using normal phase Silica gel (200-400 mesh, LOBA

    Chemical) column chromatography, employing a step gradient of increasing

    polarity from hexane to diethyl ether to methanol (100%H; 80%H:20%DEE;

    60%H:40%DEE; 40%H:60%DEE; 20%H:80%DEE; 100%DEE;

    80%DEE:20%M; 60%DEE:40%M; 40%DEE:60%M; 20%DEE:80%M;

    100%M). The eleven fractions eluted were collected individually, concentrated

    through evaporation and the antibacterial potentialities of these fractions were

    determined against human bacterial pathogens using 50 µg/disc

    concentrations in triplicates. The obtained fractions were spotted on the TLC

    plates to check the purity of the eluted fractions. The fractions with similar Rf

    values were combined and eluted again in column chromatography.

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    The primary column pooled fractions of 20%H:80%DE, 100%DEE,

    80%DEE:20%M were further fractionated using normal phase Silica gel (200-

    400 mesh, LOBA Chemical) column chromatography, employing a step

    gradient of increasing polarity from diethyl ether to Dichloromethane

    (90%DEE:10%DCM, 80%DEE:20%DCM, 70%DEE:30%DCM,

    60%DEE:40%DCM, 50%DEE:50%DCM, 40%DEE:60%DCM,

    30%DEE:70%DCM, 20%DEE:80%DCM, 10%DEE:90%DCM, 100%DCM).

    The ten eluted fractions were collected separately, concentrated through

    evaporation and assayed for antibacterial activity using 25 µg/disc

    concentrations. The obtained active fractions were spotted in TLC plates to

    check the purity of the active fraction.

    HPLC, IR, NMR and MS

    The active column fraction 90% DEE: 10% DCM which showed single

    band in TLC was subjected to Reverse phase Semi preparative HPLC (HPLC

    Shimadzu Class-Vp) to further assess the purity and to collect the pure

    fractions. HPLC was carried out at Indian Institute of Crop Processing

    Technology, Thanjavur. Infrared spectrum (for functional group analysis) of

    the active column fraction (90% DEE: 10% DCM) was recorded using FTIR

    instrument. Infrared spectrum of the active compound was obtained with

    Shimadzu spectrometer at Sankara Lingam Bhuvaneswari collage of

    Pharmacy, Sivakasi. 1H NMR, 13C (Nuclear Magnetic Resonance (NMR)

    spectra were recorded on a Brucker Advance III 500 MHz instrument at Indian

    Institute of Technology, Chennai. Molecular spectrometry was used to predict

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    molecular weight of potential bioactive compound. Molecular spectrometry

    was analyzed in Finnigan MAT 8230 instrument at Indian Institute of

    Technology, Chennai. An attempt was made with the available data to

    elucidate the molecular formula and structural characteristics of the active

    compound.

    RESULTS

    Partitioning

    The bioassay guided partitioning of the crude diethyl ether extract

    indicated wide spectrum activity in ethyl acetate phase and the zone of

    inhibition ranged from 6 to 10 mm (Table 6.1) The butanol phase showed

    antibacterial activity against 80% of the tested bacterial pathogens with an

    inhibition zone range of 1 to 7 mm. The water phase showed very low

    antibacterial activity (against 20% of bacterial pathogens). The results

    indicated the non polar nature of the active fraction.

    Column fractionation

    Among 11 primary column fractions, the fraction 6 exhibited broad

    spectrum of antibacterial activity against human pathogens (Table 6