2.1. Features of marine environment
The intensive work on the marine life resulted production of thousands of bioactive substances from marine organisms. (Ausubel et al., 2010). This great diversity of the underwater life is a huge resource of compounds for the treatment of various diseases. This remarkable yield of novel compounds from marine sources, with a wide range of bioactivities, from a tiny subset of the enormous diversity of life in the marine environment, indicates that an increased effort to find drugs from marine macro-organisms and microorganisms is likely to provide thousands of new compounds (Hill and Fenical, 2010).
Over 5000 novel compounds have been isolated from shallow waters to 900-m depths of the sea (Somnath and Ghosh, 2010). A total of 36 living phyla are known, of which 34 are recorded in marine environments (Dhinakaran et al., 2014).
2.3. Marine and marine-derived fungi
2.3.1. Fungi from the marine habitats and organisms
The presence and the association of fungi with marine organisms has been reported and discussed for over a century (Murray, 1893; Church 1893). An important investigation on fungi from the marine environment was carried out by Barghoorn and Linder (1944). Since then, the number of fungal species described from marine environments and the rate at which they are currently being described indicates that the marine fungal community is larger than expected (Jones, 2011). However, the understanding of marine fungal communities is still limited. Fungal communities in the marine environments is estimated to be comprised of 1.5 million species, of which less than 10% of are described (Hawksworth, 2001; Amend et al., 2012).
2.3.2. Ecological roles and effects of fungi in the marine environment
The physical factors that influence the marine fungi are a) salinity and pH, b) low water potential, c) high concentrations of sodium ions, d) low temperature, e) oligotrophic nutrient conditions and f) high hydrostatic pressure, the last three parameters being unique to the deep-sea environment (Raghukumar, 2008).
2.3.3. Distribution of marine-derived fungi
Sponges have yielded the greatest taxonomic diversity. The distribution of all compounds reported from marine derived fungi is shown as a function of the fungal source (Fig. 1 & Fig. 2). Other represents sources that were too small to represent a significant number of metabolites (Bugni and Ireland, 2004).
Fig. 1. The number of distinct fungal genera based on the marine source (Bugni and Ireland, 2004).
Fig. 2. Distribution of total and new compounds from fungi isolated from marine resources (after Bugni and Ireland, 2004).
2.3.4. Sessile marine animals-associated fungi
Many cases where fungi cause disease and even death and extinction of many marine organisms have been listed Fisher et al. (2012). Marhaver et al., (2013) stated that survival of planulae larvae of the coral Montastrea faveolata was due to their proximity to adults of other taxa and due to the mortality caused by the activity of host-specific detrimental microorganisms.
Fungi are known as resident and functional components of sessile marine animal microbes. The extent of fungi diversity and function has only begun to be revealed and determining of their contribution and effects on sessile marine animals is a matter of time (Marhaver et al., 2013).
2.3.5. Fungi in hard corals
The phylum Cnidaria, that contains about 10,000 species (Zhang, 2011) is the most widely studied with regard to fungal occurrence. While the presence of fungi in coral hosts is acknowledged in the literature (Bentis et al., 2000; Golubic et al., 2005).
The majority of studies on fungi associated with corals have focused on either parasitic or opportunistic interactions (Wegley et al., 2007). The distribution of all compounds reported from marine derived fungi is shown as a function of the fungal source. Other represents sources that were too small to represent a significant number of metabolites (Yarden, 2014).
The high diversity of microorganisms including fungi comprise complex assemblage called the coral holobiont (Rohwer et al., 2002; Knowlton and Rohwer, 2003).
Although the association between corals and fungi has been widely reported (Freiwald et al., 1997), very little is known about their identity or the nature of their interaction with the holobiont. Fungi were believed to be parasitic to the coral itself (Kendrick et al., 1982) or associated with endolithic symbiotic algae (Priess et al., 2000).
The recent theories represent coral fungi as symbionts that protect the holobiont from infection and disease (Rohwer et al., 2002; Reshef et al., 2006; Shnit Orland and Kushmaro, 2009), or recycle nitrogen molecules for uptake by the symbiont zooxanthellae (Wegley et al., 2007). Alternatively, coral fungi may act as mutualist, commensalist or parasite based on environmental conditions and overall coral health (Golubic et al., 2005; Muscatine et al., 2005; Wegley et al., 2007; Lesser et al., 2007; Thurber et al., 2009).
Amend et al. (2012) investigated the relationship between the Symbiodinum, the environment and fungi associated with Acropora hyacinthus a reef building, tropical coral in Ofu Island in American Samoa.
Phylogenetic analysis of fungi associated with coral- ribosomal DNA showed a high diversity of Basidiomycetes and Ascomycetes, with several clades separated from the well-known fungi by long and well-supported branches. There are more phylogenetically diverse fungal communities in the warmer pool coral colonies than the colder pool colonies and they also have statistically significant species
Messenger RNA sequenced from a subset of these same colonies contained an abundance of transcripts involved in metabolism of complex biological molecule. The concurrence between the taxonomic diversity found in the DNA and RNA analysis indicates a metabolically active and diverse resident marine fungal community in corals (Amend et al., 2012).
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Endolithic algae and fungi that penetrate coral skeleton are of particular interest. Low light intensities (Halldal, 1968) and low and fluctuating oxygen pressures (Shashar and Stambler, 1992) within coral skeleton render this environment an ecological extreme, which only a few specialized organisms can endure.
The presence of endolithic fungi in mollusk shells has been known for more than a century (Bornet and Flahault, 1889), and a number of papers discuss their taxonomic identity and ecological significance (e.g. Pulicek 1983, Porter and Lingle 1992, Hook and Golubic 1993).
Less information is available on identity, diversity and ecological roles of fungi in corals. Most reports on coral diseases (e.g. Antonius, 1981; Goldberg and Makemson, 1981; Rutzler et al. 1983; Goldberg et al., 1984) do not include fungi among coral pathogens. It is assumed that their effect on the host is negligible, or that the fungi in corals are saprophytes that exploit dead organic matter incorporated in coral skeletons by the coral or produced by endolithic algae and cyanobacteria (Kendrick et al., 1982).
A successful isolation and culturing of higher, ascomycotic and basidiomycotic fungi from the skeletons of Atlantic and Pacific hermatypic allowed the first identification of fungal genera and species that were likely candidates for the known and widespread coral borers. Kendrick et al. (1982) cultured 20 fungal taxa isolated from coral reefs, documenting the presence and viability of fungal propagules in this marine environment. A similar approach was used by Raghukumar and Raghukumar (1991), who reported fungi associated with coral necrosis. Both studies reported distribution of fungi inside coral skeleton but did not establish fungal pathogenicity.
Bak and Laane (1987) reported dark discoloration and banding inside coral skeleton caused by fungi. They also called attention to a possible active interaction between corals and fungi.
Le Campion et al.(1995) studied the growth of endohthic algae and fungi that occurs within the polyp zone of the hermatypic coral Porites lobata, parallel and concurrent with skeletogenesis in French Polynesia. They discussed the spatial and functional interrelationship between endohthic fungi, algae and corals. In addition, they documented a defence response by corals to fungal invasion which leaves a permanent mark in coral skeleton. Finally, differences in early diagnosis between intact and affected coral skeleton are reported. They stated that Fungi may be opportunistic pathogens in corals under environmental stress. Their activity, recorded and preserved in the coral skeleton, provides information on changes in past conditions of coral growth.
Fungi also cause coral reef disease: Aspergillosis, which causes purple blotches on several species of sea fans, is caused by Aspergillus sydowii, a member of a large group of terrestrial fungi that also trigger mold allergies and other infections in humans (Geisner et al., 1998).
The damage caused by reactive oxygen species is considered a contributing factor to AD. Three fungi (FC1, FC2 and FC3) were isolated from the soft corals; Sinularia sp. and Lobophyton sp. The antioxidant activities (DPPH and Xanthine Oxidase assays), (AChE) inhibitory activity and antimicrobial activity for twelve different fungal extracts were evaluated.
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Didha Andidni Putria (2015), isolated 15 fungal symbionts that were capable of growth inhibition of pathogenic fungi from soft coral Sinularia sp. from Panjang island of the North Java Sea. These fungi were screened against pathogenic fungi Candida albicans and Aspergillus using overlay method. Phytochemical tests showed that phenolic, triterpenoid and flavanoid compounds were detected within fungal extract. Molecular identification using 18S rRNA gene indicated that the active fungi was closely related to Aspergillus unguis.
Arumugam et al. (2015) isolated a piezotolerant fungus Nigrospora sp. from deep sea environment and cultured it under submerged fermentation and extracted secondary metabolites with potent antimicrobial and anticancer activities with immediate application to cosmetics and pharmaceutical industries.
El- Hady et al. (2015), isolated the fungus Aspergillus unguis from the marine Sponge Agelas sp., Red Sea, Egypt.
In general, most studies conducted on fungi in corals focused on parasitic or opportunistic interactions. Even though, the majority of these describe either the fungal species or potential detrimental outcomes of the interaction (Wegley et al., 2007).
2.3.6. Fungi from sponge
Fungi along with other microorganisms are associated with sponges where microorganisms form about 40-60% of the sponge���s biomass (Hentschel et al., 2012). Morrison-Gardnier (2002) isolated over a hundred fungal strains from marine sponges during the last 20 years. Holler et al. (2000) isolated fungi from 16 species of sponges from different areas. A total of 85 fungal taxa have been isolated from the sponge Ircinia wariabilis in the Mediterranean Sea.
The specificity of fungal communities in different sponges has been provided by the sponges Suberites zeteki and Mycale armata (Gao et al., 2008; Li and Wang, 2009).
Assessment of fungal diseases in sponges is more difficult than those of hard corals due to the complexity of microorganism associated with sponges (Webster and Tylor, 2012). However, the potential of a sponge to be symptomless carrier of coral-disease-causing agent has strains of A. syndowii from Spongia obsucra (Ein-Gil et al., 2009).
2.3.7. Fungi from marine sediments
Most fungi occurring in the marine sediment are facultative marine fungi with terrestrial origins. It is not known yet if sediment fungi can survive the harsh environmental conditions in the ocean sediment, while playing an active role in this unique ecological niche. in the marine sediment collected from St. Helena Bay, on the west coast of the Western Cape, South Africa, 59 fungal isolates were obtained and identified to at least genus level using morphological and molecular methods.
A series of tests have been carried out to portray the physical and physicochemical traits of the isolates. Results indicated that the isolates survived and grw in the natural conditions present in this environment. Extracellular cellulase produced by the filamentous fungal isolates indicates their probable role in detrital decay processes and therefore the carbon cycle on the ocean bed. Also, denitrification patterns were observed when isolates were grown in liquid media amended with NaNO2, NaNO3, and NH4SO4, implicating that these fungi have the potential to play an active role in denitrification, co-denitrification, and ammonification phases of nitrogen cycles occurring in the marine sediments (Diversity and characterization of culturable fungi from marine sediment collected from St. Helena Bay, South Africa (Mouton M,2012).
Zhang et al. (2015) evaluated the diversity and distribution of fungal communities in marine sediments of Kongsfjorden (Svalbard, High Arctic) using 454 pyrosequencing with fungal-specific primers targeting the internal transcribed spacer (ITS) region of the ribosomal rRNA gene. Fungal communities in the sediments presented high diversity with 42,219 reads belonging to 113 operational taxonomic units (OTUs). Of which, 62 belonged to the Ascomycota, 26 to Basidiomycota, 2 to Chytridiomycota, 1 to Zygomycota, 1 to Glomeromycota, and 21 to unknown fungi. The major known orders included Hypocreales and Saccharomycetales. The common fungal genera were Pichia, Fusarium, Alternaria, and Malassezia. Most fungi collected from Arctic sediments may have the terrestrial origin from different basins in Kongsfjorden (i.e., inner basin, central basin, and outer basin) harbor different sedimentary fungal communities. These results suggest the presence of diverse fungal communities in the Arctic marine sediments, which may serve as a useful community model for further ecological and evolutionary study of fungi in the area (Zhang et al, 2015).
Surveys on the molecular diversity of the micro-eukaryotic community have proved that fungi are essential component in a large number of marine habitats. Molecular tools proved that fungi are present in large number of marine habitats such as deep-sea habitats, pelagic waters, coastal regions, hydrothermal vent ecosystem, anoxic habitats, and ice-cold regions. Majority of the environmental phylotypes could be grouped as novel clades within Ascomycota, Basidiomycota, and Chytridiomycota or as basal fungal lineages. Raghukumar C, 2013) grouped the deep-branching novel environmental clusters within Ascomycota as the Pezizomycotina clone group, deep-sea fungal group-I, and soil clone group-I, within Basidiomycota as the hydrothermal and/or anaerobic fungal group, and within Chytridiomycota as Cryptomycota or the Rozella clade. However, they identified the basal true marine environmental cluster as most of the clusters include representatives from terrestrial regions.
Fungi have also been reported in several deep sea environments (Nagahama et al., 2006), including hydrocasts near hydrothermal plumes from the Mid-Atlantic Ridge (Gadanho and Sampaio, 2005) and in Pacific sea-floor sediments (Nagahama et al., 2010). Fungi from both of these habitats were dominated by unicellular forms commonly designated as yeasts. Presence of fungi in the deep sea sediments of the Central Indian Basin was reported by direct detection and immune-fluorescence techniques (Damare et al., 2006). They were also isolated from the deep seas sediments by various isolation techniques.
The importance, ecological roles and diversity of marine fungi have recently been reported in deep subsurface sediments using molecular traits. Fungi in the deep marine subsurface may be specifically adapted to life in the deep biosphere, but this can only be demonstrated using culture-based analyses. In this study, culturable fungal communities from a record-depth sediment core sampled from the Canterbury basin (New Zealand) was investigated with the aim to reveal endemic or ubiquist adapted isolates playing significant ecological role(s). About 200 filamentous fungi (68%) and yeasts (32%) were recorded. Fungal isolates were belonging to Ascomycota and Basidiomycota phyla, including 21 genera. Screening for genes involved in secondary metabolite synthesis also revealed their bioactive compounds synthesis potential. These results provide evidence that deep subsurface fungal communities are able to survive, adapt, grow and interact with other microbial communities and highlight the deep sediment habitat as another ecological niche for fungi (Vanessa R��dou,et al., 2015).
2.3.8. Fungi in mangroves
The study of marine fungi inhabiting mangroves was initiated 50 years ago in Australia (Cribb and Cribb 1955, 1956 Kohlmeyer and Kohlmeyer 1979). Ecological and taxonomic studies have been performed since the 1980s in the Pacific and Indian Oceans (Hyde, 1986; Jones and Tan, 1987; Hyde and Jones, 1989; Hyde et al., 1990; Alias et al., 1995; Poonyth et al., 1999; Maria and Sridhar, 2004).
Until recently, few ecological studies on manglicolous fungi were conducted. Recent studies on inertial fungi in mangrove have provided information on frequency of occurrence, vertical zonation, host and substratum specificity, succession, and seasonal occurrence of fungi (Aleem, 1980; Jones et al., 1988; Poonyth et al., 1999). Considerable effort has been spent investigating the frequency of occurrence of manglicolous fungi (Jones and Tan, 1987; Borse, 1988; Hyde and Jones, 1989; Jones and Kuthubutheen, 1989; Tan et al., 1989; Tan and Leong, 1992).
A detailed investigation of fungi on mangroves of west coast of India was carried out by Patil and Borse (1985) and Chinnaraj (1993). However vast tracts of mangroves on the east coast remained virtually undiscovered except for the studies of Ravikumar and Vittal (1996).
Adaptation and activity of terrestrial fungi under marine/mangrove ecosystem as facultative or in-dwellers or residents were investigated (Raghukumar and Raghukumar, 1998: Chandralata, 999).
Terrestrial fungi are common in mangrove water and mud (Garg, 1983), mangrove leaves (Raghukumar et al., 1995), wood (Aleem, 1980), standing senescent stems (Sadaba et al., 1995), decomposing mangrove palm (Nypa fruticans) (Pilantanapak et al., 2005).
Terrestrial fungi in deep sea region of Arabian Sea were recovered by Raghukumar and Raghukumar (1998). Sampling of the leaf litter from the Nethravathi mangroves, India revealed the occurrence of many freshwater Hyphomycetes (Sridhar and Kaveriappa, 1988).
Borse (2002) reported that the distribution and substratum range of 166 species of marine fungi recorded so far from India on animal substratum, driftwood, intertidal wood, algae, mangroves, sea grasses, salt marsh plants and propagules in the sea foams samples.
The richness and diversity of filamentous fungi on woody litter of mangrove along the West coast of India have been studied (Ananda and Sridhar, 2002; Maria and Sridhar, 2002). Most fungi recorded from marine sediments are terrestrial in origin (Vrijmoed, 1990).
In the Red Sea, Aleem (1978) recorded Corollospora pulchella and Periconia prolifica. From the Red Sea coast of Saudi Arabia Schatz (1985) described Adomia avicenniae on pneumatophores of Avicennia marina.
A total of 36 marine fungi species were isolated from Safaga mangrove on the Egyptian Red Sea, of which 18 species on decayed attached mangrove wood of A. marina (El-Sharouny et al. (1998). Other 18 fungi on submerged decayed leaves of A. marina and algal thalli collected from Safaga mangroves (El-Sharouny et al. (1999).
Abdel-Wahab (2000) recorded 25 fungi on intertidal wood of A. marina collected from three mangrove stands, namely Sharm El-Sheikh, Abu-Mingar and Safaga located on the Red Sea coast of Egypt. Three new species, namely Halosarpheia unicellularis, Swampomyces aegyptiacus, and S. clavatispora were published from Red Sea mangroves in Egypt (, b).
Abdel-Wahab et al., (2001a) examined the diversity of marine fungi on intertidal decayed wood of Avicennia marina and on decayed prop roots of Rhizophora mucronata in the mangrove stands located in the southern part of the Egyptian Red Sea coast.
Diversity of marine fungi in Red Sea mangroves in Egypt was assessed, and fungi dominating the communities were recorded and compared with those from other mangroves in subtropical and tropical regions (Abdel-Wahab (2005). Analysis of the decayed wood samples of A. marina collected from six mangrove stands from Egyptian the Red Sea coast resulted in the record of 39 fungal species were identified of which, 19 are new records for the area.
2.4. Fungal diseases of marine animals
Fungal diseases can act as major limitors of natural and cultured populations of marine animals (Alderman and Polglase, 1986). Mycopathogens of aquatic animals have become the focus of considerable attention because of the high occurrences of fungal diseases in wild populations and aquaculture (Polglase et al., 1986; Noga, 1990).
Marine fungal pathogens are predominantly straminipilous organisms (oomycetes), but also include some mitosporic fungi. The oomycetes are probably most important and include several genera (e.g. Aphanomyces, Halocrusticida (.Atkinsiella), Haliphthoros, Lagenidium, Leptolegnia, Saprolegnia, Sirolpidium) comprising species that are notoriously destructive pathogens (Rand, 1996). Most of these have been described from bivalve molluscs, or crustaceans (Noga, 1990).
Thraustochytrids and labyrinthulids have also been reported to cause infections in many marine animals, including abalones Haliotis spp., the large nudibranch Tritonia diomedea, and the lesser octopus Eledone cirrhosa (Bower, 1986; McLean and Porter, 1987; Hyde et al., 1998).
The most important mitosporic fungal pathogens are Fusarium species (e.g. F. solani) which have been reported to be associated with shell disease of marine crustaceans (Lightner, 1988), and mycotic infections in hermit crabs (Smolowitz et al., 1992) and lobsters (Stewart, 1984).
Other mitosporic fungal pathogens include an unnamed Scolecobasidium which causes infection among massive coral species from the Andaman Islands (Raghukumar and Raghukumar, 1991), Ochroconis humicola which causes ulcerative lesions in devil stinger (Inimicus japonicus) cultured in Japan (Wada et al., 1995), and Lasiodiplodia theobromae which was isolated from an infection in a juvenile boring clam (Tridacna crocea) cultured in Australia (Norton et al., 1994).
Most marine fungal infections, once established in an individual, are often fatal and difficult to treat. This indicates that these fungi will continue to be problematic pathogens of marine animals (Noga, 1990).
Occurrence of fungi in healthy and diseased corals has been widely reported from all over the world. Raghukumar and Raghukumar,reported the presence of Scolecobasidium from Porites, Montipora, Goniopora ad Goniastra. This fungal parasite was found to cause nerotic patches on these five of the seven corals examined from Andaman Island. A detailed investigation for the presence of fungi in healthy, partially dead and bleached corals has been carried out (Hyde et al.,1998).
Several species of fungi have been isolated from healthy, bleached and dead corals. The highest frequency of fungi with most genera of terrestrial origin was on the partially dead corals. Of the most frequently encountered species were, Curvularia lunata, Aspergillus, and Cladosporium. Scanning electron photomicrographs revealed the presence of fungi within the calcium carbonate skeleton and around the polyp. It was then concluded that fungi are regular skeletal components of healthy, partially dead and diseased corals (Hyde et al., 1998).
2.5. Marine organisms as a source of bioactive substances
The ocean is considered to be a source of potential drugs and natural products from the ocean have been considered by biologists and chemists for the last decades which results in the extraction of thousands of marine natural products. Most the extracted compounds from marine source have biological activity. (Mayer and Hamann, 2005; Blunt et al., 2007; Somnath and Ghosh, 2010).
Marine organisms can elaborate pharmaceutically useful compounds as well as toxic ones. One of the most important common contribution of marine natural products has been the isolation and identification of toxins responsible for seafood poisoning. Most marine toxins are produced by microorganisms such as dinoflagellates or marine bacteria and may pass through several levels of the food chain. The identification of marine toxins has been one of the most challenging areas of chemistry of marine natural products (Mayer and Hamann, 2005; Blunt et al., 2007; Somnath and Ghosh, 2010).
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The job of chemists of the marine natural products for the past decades has been the search for potential pharmaceuticals. It is difficult to distinct a particular bioactive molecule that is designed to take a part in medicine (Mayer and Hamann, 2005; Blunt et al., 2007; Somnath and Ghosh, 2010).
Marine organisms produce some of the most cytotoxic compounds ever discovered, but the amounts of these compounds are too small to to provide enough material for drug development studies (Mayer and Hamann, 2005; Blunt et al., 2007; Somnath and Ghosh, 2010).
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Marine environment offers various biosynthetic conditions to organisms that live in it. Marine organisms generally live in symbiotic association. The pathway of transfer of nutrients between symbiotic partners is of much importance and raises questions about the real origin of metabolites produced by association. A recent trend in marine natural products chemistry is the study of symbiosis. Biosynthesis of bioactive marine natural products provides many challenging problems (Mayer and Hamann, 2005; Blunt et al., 2007; Somnath and Ghosh, 2010).
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Marine organisms are a rich source of structurally novel and biologically active metabolites. Recently, studies have suggested that some bioactive compounds isolated from marine organisms have been shown to have anti-cancer, antihelmintic, antibacterial, anti-fungal, antiviral, antimalarial, anticoagulant, antiprotozoal, antituberculosis, or anti-inflammatory and other pharmacological activities (Mayer and Hamann, 2005; Blunt et al., 2007; Somnath and Ghosh, 2010).
To date, several chemical compounds from marine organisms have been isolated and are under investigation (Febles et al., 1995; Siddhanta et al., 1997).
Marine organisms including sponges, sponge-microbe symbiotic association, gorgonian, actinomycetes, and soft coral have been widely explored for potential anticancer agents (Bhatnagar and Kim, 2010).
2.2.1. Marine bioactive substances from sponges
Marine sponges are the prolific source of bioactive molecules with novel chemical structure. Sponges- the most primitive sessile filter feeders-produce these compounds as secondary metabolites as part of chemical defense against predators, space competitors and fouling since they lack physical defense mechanisms. Sponges, the most diverse marine invertebrates have evolved antagonistic effects against other invading organisms, which involves the production of the secondary metabolites (Wah et al., 2006).
Sponges, in particular, are responsible for a large number of these compounds, which exhibit a wide range of activities including antitumor. For example, 13-Deoxytedanolide is a potent antitumor macrolide isolated from the marine sponge Mycale adhaerens (Nishimura et al., 2005); as an antiviral: sponge aqueous extract was tested for anti-herpetic, anti-adenovirus and anti-rotavirus activities. (Da Silva et al., 2006), as antibacterial: antibacterial extraction from sponges were used against certified strains of bacteria (Staphylococcus aureus and Escherichia coli) and yeast (Candida albicans) giving 80% positive results (Galeano and Mart��nez, 2007) and antifungal activity (Concepcion et al., 1994).
Halichondrin B and structurally related congeners were isolated from several species of sponges, including Halichondria okadai from the waters of Japan, an Axinella sp. from the western Pacific, Phakellia carteri from the eastern Indian Ocean and Lisodendoryx sp. from deep water off New Zealand's coast (Piel,2004).
The results of initial cytotoxicity screens generated enough excitement about halichondrin B's potential as an anticancer agent that a massive collection program was conducted. Later in these studies, it was discovered that this sponge could be aquacultured in less than 30 feet of water with good success, and with comparable amounts of halichondrin B present (Piel,2004).
In reality, many recent studies have revealed that the sponges themselves likely contribute little chemistry to the assembly of these compounds; rather, the sponges are basically repositories for many species of symbiotic bacteria and other microflora that actually perform the biosynthesis of the isolated chemical species. In fact, the microfloral content of many sponges constitutes more than half the sponge's dry weight (Piel, 2004).
Back to halichondrin: Sub-nanomolar (~ parts-per-billion) in vitro potency against a variety of cancer cell lines, including non-small cell lung cancer, marked halichondrin B as a candidate for clinical testing (Newman, and Cragg, 2004).
The E7389 is the common name given to a synthesized derivative of the sponge isolate halichondrin B (Newman and Cragg, 2006b). E7389 exhibits an anticancer profile similar to that of the parent, but E7389's greatly simplified structure (the green half of halichondrin B has been deleted) renders it a far more attractive candidate for drug development.
2.2.2. Marine bioactive substances from worms
Few studies were done on marine worms especially polychaetes. Marine worms dwell in sediments, indicating the requirement of antimicrobial strategy for their survival. Perinerin, Arenicins-1, 2 and Hedistin have been isolated from polychaete and echiuroid worms. Arenicin-1 and -2 were isolated from coelomoycytes of the polychaete Arenicola marina (Ovchinnikova et al., 2004).
Perinerin isolated from homogenates of the polychaete Perinereis aibuhitensis, is a highly cationic, hydrophobic peptide (Pan et al., 2004). It had an antifungal and antibacterial activity against Gram-negative and Gram���positive bacteria. Organobromine compounds were produced naturally by marine creatures (sponges, corals, sea slugs, tunicates, sea fans), also brominated compounds such as brominated indoles, 2,3,4-tribromopyrrole and brominated phenols were produced naturally, by common polychaete worms (Gribble, 2000). The antimicrobial activities of dominant fatty acids were assessed against marine pathogenic bacteria by Benkendorff et al. (2005).
Ibrahim and Abd El-Naby (2010) collected five marine polychaete species isolated from seawater, Alexandria, Egypt, which were taxonomically identified and then investigated as a source of natural products which can be used as antibacterial and antifungal of human and fish pathogens. Three of these species were from family Nerididae; genus Nereis (Nereis falsa), genus Perinereis (Perinereis nuntia typica) and genus Pseudonereis (Pseudonereis anomala); one species from family Oenonidae; genus Halla A. (Halla parthenopeia), and finally one species was from family Serpulidae; genus Hydroides (Hydroides elegans). However, the gas liquid chromatography mass spectrometer of H. parthenopeia and H. elegans extracts was determined and the main constituents detected were organic acids and their derivatives.
2.2.3. Marine-derived compounds from bryozoan
Bryostatins are a group of macrolide lactones first isolated in the 1960s by George Pettit from extracts of a species of bryozoan, Bugula neritina (Shimek, 2003) Cragg et al., 2005). Bryostatins were among the first marine isolates to indicate the importance of bioactive compounds from bryozoans as anticancer. Their promising initial responses in early anticancer screens led to large-scale collections to compensate for the low amount of material taken from each organism (< 10 milligrams, at best, from a kilogram of B. neritina). This level of collection was not sustainable, aquaculturing efforts that led to successes for both mariculture and aquaculture of bryozoans (Shimek, 2003).
The wide geographical range of Bugula neritina and the common observation that many colonies cannot yield bryostatin led to a conclusion that a commensal microorganism, Candidatus Endobugula neritina and not the bryozoan itself, was the one who produces bryostatins (Shimek, 2003).
This observation have some significance answers to the question of large-scale bryostatin manufacture if they are to be used as anticancer drug in the commercial scale. in the meantime, it is difficult to identify the suitable conditions of cultivation and harvesting of whole marine invertebrates such as Bugula neritina on a scale commensurate with commercial needs. However, using microorganisms as source of bioactive substances has had a long and successful history in the pharmaceutical industry. Therefore, culturing Candidatus Endobugula neritina, or transferring its bryostatin-producing gene cluster into a well-behaved surrogate microbe, may become viable options for the commercial manufacture of bryostatin 1 (Shimek, 2003). However, the mechanism-of-action of Bryostatin's is yet again different from those discussed with the other anticancer compounds (Cragg et al., 2005).
2.2.4. Marine bioactive substances from mangrove
Mangrove ecosystems is the second most productive marine ecosystem. Many mangrove species have been used as antiviral. Bark extract of Rhizophora mucronata and leaf extract of Bruigiera cylindrica were highly effective against all viruses (Premanathan et al., 1996).
Azuma et al. (2002) investigated the chemistry of odor of a number of mangrove species. A total of 61 compounds were found, these were fatty acid and carotenoid derivatives or terpenoids. The chemical profiles of individual species appear to be unique ranging from only two compounds in the mangrove Kandelia candel to 25 in mangrove Nypah fruticans. Synergistic action needs also to be taken into consideration, e.g. berberine isolated from rhizomes of Berberis aristata has antimicrobial and antifungal activity and its activity increases when used in conjunction with santonin (Singh et al., 2001).
2.2.5. Marine bioactive substances from algae
Investigations on the bioactive substances during the last three decades have been focused mainly on screening for the biologically active compounds in seaweeds against various human pathogenic viruses, bacteria and fungi (Vidyavathi and Sridhar, 1991; Premnathan et al., 1992; Kamat et al., 1992; Robles-Centeno et al., 1996; Sastry and Rao, 1994).
Many bioactive and pharmacologically important compounds such as alginate, carrageen and agar as phycocolloids have been extracted from seaweeds and used in medicine and pharmacy (Siddhanta et al., 1997). Fatty acids are isolated from micro algae that exhibited antibacterial activity (Kellam et al., 1998).
There are several reports on substances extracted from seaweeds with a wide range of biological activities, such as antibacterial (Nair et al., 2007), antivirals (Richards et al., 1978), antitumorals (Espeche et al., 1984), anticoagulant (Marechal et al., 2004) and antifouling (Newman et al., 2003).
The extracts and active constituents of different seaweeds have been shown to exhibit antibacterial activity in vitro against Gram-positive and Gram-negative bacteria. The production of antimicrobial activities was an indicator of the ability of the seaweeds to produce bioactive secondary metabolites (del Val et al., 2001).
2.2.6. Bioactive substances from soft corals
Soft-bodied corals, are unlimited sources of many secondary metabolites that display important biological activities. Unfortunately, the subset of coral metabolites that exhibit significant anticancer activity on the order of the compounds described above is minimal at present (Taglialatela-Scafati, 2002).
Soft corals contain a large variety of sesqui-terpenoids and di-terpenoids of which many are found to be toxic (Groweiss et al., 1985).
The crude extracts from ten soft corals inhabiting the Red Sea (Lopophytum pauciliforum, Dendronephthea, Sarcophyton gracile, Sarcophyton glaucum, Sinularia gardineiri, Nephthea pacifica, Sarcophyton acutum, Lopophytum sp., Sarcophyton acutum and Xenia macrospiculata) were screened for the antimicrobial activity.
These extracts exhibited appreciable but variable antimicrobial activity against different potentially pathogenic fish and human bacterial and fungal pathogenic strains examined (Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 8739, Vibrio damsela, Vibrio fulvilalis, Bacillus cereus, Bacillus cereus 1318, Streptococcus faecalis, Escherichia coli Fusarium oxysporum, Rhizoctonia solani, Penicillium oxalicum and Aspergillus niger) using well cut – diffusion technique.
These results confirmed that the absolute activity units (AU) of the ethanolic crude extract ranged from 1.4 to 25.0 for Sarcophyton acutum and Lopophytum pauciliforum, respectively (Ibrahim et al., 2013).
The evolutionary success of soft corals in areas of high levels of predation may be due to their production of significant quantity of secondary metabolites, especially terpenes (Sammarco and Coll, 1992). Many soft corals show predator deterrence activity (Pawlik et al., 1987; Van Alstyne et al., 1994).
Pseudopterolide, (diterpene) from the gorgonian Pseudopterogorgia acerosa exhibits unusual cytotoxic properties (Banduraga et al., 1982).
Soft corals of the genus Sinularia was found to be rich source of bioactive secondary metabolites. Four new substances have been isolated from Sinularia sp. collected off the northeastern Taiwan coast. The soft corals of the genus Nephthea are also rich in terpenoids and steroids (Chih-Hua Chao et al., 2006).
Gamal et al. (2004) isolated five new bioactive substances from the methylene chloride extracts of the soft coral Nephthea armata from Taiwan because its extract showed significant cytotoxicity to human lung adenocarcinoma and human colon adenocarcinoma as well as mouse lymphocytic leukemia.
The soft corals of the genus Xenia are rich in terpenoids and steroids. Six new sesquiterpenoids, xenitorins A-F were isolated from the methylene chloride extracts of the soft coral of Xenia peurtogalera. The structures were elucidated by 1D and 2D NMR spectral analysis and their cytotoxicity was determined against selected cancer cells (Yih Duh et al., 2002).
Recently, Lobophytum cristagally another soft coral species was reported to have cembrane diterpene (Coval et al., 1996; Matthee et al., 1998). Japanese soft coral Lemnalia sp is reported to provide Lemnalol which is known for anti-tumor activity (Kikuchi et al., 1983).
The Formosan soft coral Cespitularia hypotentaculata (family: Xeniidae) was studied because its CH2Cl2 extract showed significant cytotoxicity to human lung and mouse. The fractionation resulted in the isolation of new four cytotoxic diterpenes, cespitularins A-D, cesputularin, cespitularins F-H and cespitularane (Yih Duh et al., 2002).
The Eleutherobin is one of the more promising candidates from this small pool. It was originally isolated from the octocoral Eleutherobia sp. in Australian waters, and its preliminary anticancer screens were encouraging (Taglialatela-Scafati, 2002).
The verification of the medicinal value of some soft corals by modern scientific work emphasized the importance of the compounds isolated from corals.
The Sarcophytolide is a new neuroprotective compound isolated from the soft coral Sarcophyton glaucum (Badria et al., 1998). Moreover, two new bicyclic cembranolides that were found to be cytotoxic toward an MCF7 tumor cell line were isolated from a new Sarcophyton species (Gross et al., 2004).
A new norcembrane, designated sinularectin 3, was isolated from the Kenyan soft coral Sinularia erecta. Sinularectin is a chlorinated highly oxygenated norcembrane with an unprecedented functionalisation of the cembrane isopropyl group (Rudi et al., 2006).
The furanocembranoids 2, 4, and 5 that show good antiproliferative activities against the cell lines, and weak cytotoxic effects on HeLa cells, were isolated from the Soft Corals Sinularia asterolobata and Litophyton arboretum (Grote et al., 2008).
Screening of the dichloromethane fraction of the marine sponge Phyllospongia lamellosa collected from the Red Sea resulted in the isolation and characterization of new scalarane sesterterpenes (Hassan et al., 2015).
Soft corals of the genus Lobophytum, have shown diverse biological activities as anti-inflammatory, cytotoxic, and antibacterial. Hassan, (2016) investigated the biological activity of the Red Sea soft coral Lobophytum pauciflorum. In vitro cyclooxygenase inhibitory activity using COX-1 and COX-2 kits and antimicrobial screening were carried out for n-hexane, dichloromethane, ethyl acetate and methanol fractions. The isolated compounds were elucidated using different spectroscopic methods including nuclear magnetic resonance and mass spectrometry. Also n-hexane fraction was subjected to GC/MS analysis. Bioassay guided fractionation resulted in isolation and characterization of two bio-active metabolites nephthenol (2) and gorgost-5-ene-3��-ol (3) with significant in vitro antiinflammatory activity against COX-1 and COX-2 compared to Indomethacin and Celecoxib. Four other compounds were also isolated: Heptadecan-1-ol (1), palmitic acid (4), stearic acid (5) and batilol (6). The isolated compounds showed antimicrobial activity ranging from 25 ��g/ml to 50 ��g/ml against the tested microorganisms. The fatty acid constituents of the n-hexane fraction were identified by GC/MS analysis; results revealed the presence of hexadecanoic acid, methyl ester as major saturated fatty acid and 7,10-hexadecadienoic acid, methyl ester as major unsaturated fatty acid (Hassan, 2016).
Ten compounds including five sterols (1-5), three sesquiterpenes (6-8) and two fatty acid esters (9-10) have been isolated from the Red Sea soft coral Sinularia terspilli. The anti-leukemic, anti-leishmanial, antimicrobial, and antimalarial activities of the isolated compounds and some acetylated derivatives (1Ac, 3Ac, 4Ac, 5Ac) were evaluated. Compounds 4, 5, and 5Ac exhibited strong cytotoxic activity against human leukemia cell lines HL60 and K562 with IC50 values of 4.0, 2.0 and 25 nM, respectively for HL60 and 5.0, 3.0 and 4 0 nM for K562 (Rabab Mohamed, 2015).
2.2.7. Marine-derived compounds from bacteria
Marine bacteria are increasingly being recognized as a significant resource for microbial products that display antibacterial and antifungal properties (Jensen and Fenical, 1994; Riley and Wertz, 2002; Woo et al. 2002).
Marine epiphytic bacteria, associated with nutrient-rich algal surfaces and invertebrates, have also been shown to produce antibacterial secondary metabolites which inhibit the settlement of potential competitors (Bernan et al., 1997; Boyd et al., 1999; Woo et al., 2002). In addition, bacteria in bio-films on the surfaces of marine organisms have been documented to contain a higher proportion of antibiotic producing bacteria than some other marine environment (Lemos et al., 1986).
2.6. Characterization of proteinaceous compounds
The techniques used for characterizing marine bioactive substances depend upon the nature of this agent and the target for which the characterization is carried out.
Generally, the antimicrobial agents can be categorized into two main groups: compounds with proteinaceous nature (e.g. peptides, bacteriocins, enzymes and other proteins) and organic compounds without proteinaceous nature (e.g. antibiotics, alkaloids, terpenoids, steroids, siderophores, etc ���.).
The investigators involved in this field have utilized a wide variety of different. Combination of procedures of assessment of these compounds due to the extremely heterogeneous nature of proteins (Klaenhammer, 1993).
However, the characterization of proteins depends upon their purification. Illustration of their biochemical structure requires homogeneity as well as an adequate yield of proteins (Carolissen-Mackacy et al., 1997). In some cases, structural elucidation of proteins cannot be performed using standard spectral techniques (Jensen and Fenical, 1994). The sensitivity of the quantitative protein assay and antagonistic assay are important factors to bear in mind when assessing the degree of purity and specific activity (Tagg et al., 1976).
The characterization of proteins is performed through some common procedures (Yang et al., 1992; Todorov and Dicks, 2004). These involve:
(i) Precipitation using ammonium sulfate.
(ii) Purification by filtration using gel filtration, ultra-filtration, dialysis, high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC).
(iii) Extraction using organic solvents (acetone, butanol, ethanol, propanol, isopropanol, etc���.).
(iv) Determination of purity degree and molecular weight of protein using SDS-gel electrophoresis.
(v) Determination of molecular weight (mass) of protein using mass spectrometry techniques.
(vi) Determination of the amino acids composition of protein using amino acids analyzers and/or,
(vii) Elucidation of the tertiary structure of protein using nuclear magnetic resonance (NMR).
The detection and separation of the antibiotics are carried out by using TLC and HPLC techniques (El-Banna and Winkelmann, 1998; Duffy and Defago, 1999).
From several studies, physical and chemical characterizations of the antibiotics have been detected (IR-spectrum, masspectrum, H-NMR spectrum and 13C-NMR spectrum) (El-Banna and Winkelmm, 1998; Isnansetyo and Kamei, 2003).
Both types of siderophores (hydroxamates and carboxylates) can be detected by spectrophotometric tests (Calugay et al., 2004).
These techniques are capable of separating and detecting multiple siderophores present in low concentration (nM) in seawater (McCormack et al., 2003; Gledhill et al., 2004) are performed by ultra violet (UV)-spectrum, infrared (IR).