MARINE BIOACTIVE COMPOUNDS AND THEIR APPLICATIONS IN MARICULTURE

A.P. LIPTON,

Vizhinjam Research Centre of CMFRI, Vizhinjam

Introduction

Marine bioactive compounds or Marine natural products (MNPs) are organic compounds produced by microbes, sponges, seaweeds, and other marine organisms. The host organism synthesizes these compounds as non-primary or secondary metabolites to protect themselves and to maintain homeostasis in their environment. Between 1977 and 1987, around 2500 new metabolites (MNPs) were reported from marine organisms ranging from microbes to fish, which accounts for less than 1.0% of the total marine organisms. Review of literature reveals that even the seawater has bactericidal properties. This could be attributed to the production of antibiotics by planktonic algae and bacteria respectively.

In the case of aquaculture or Mariculture activities, diseases are frequently encountered in all the stages (from the egg stage onwards). Disease control using antibiotic substances has inherent limitations. Recently, the Marine Products Export Development Authority (MPEDA) of India has restricted the usage of many antibiotics in aquaculture, particularly in shrimp farming. The common problems of antibiotic use include: development of drug resistant bacteria, environmental contamination and possible residues in the tissues of fish/shellfish. Disease management using vaccines has limitation as they are specific. If the causative agent is different, the vaccine will not work. Considering the potential of marine bioactive substances, and the avenues for developing potent new drugs and other useful products, a holistic approach is required to develop fish/ shellfish therapeutics, immunostimulants and other feed additives.

Marine bacteria

Rosenfeld and Zobell (1947) demonstrated that marine bacteria produce anti-microbial substances. The first documented identification of a bioactive marine bacterial metabolite was the highly brominated pyrrole antibiotic, isolated by Burkholder and co-workers from a bacterium obtained from the surface of the Caribbean Sea grass Thalassia (Burkholder et al., 1966). Subsequently, this unique metabolite was identified by x-ray crystallographic methods, which composed of more than 70% bromine by weight (Lovell, 1966). The metabolite exhibited impressive in vitro antibiotic properties against Gram-positive bacteria, with minimum inhibitory concentration (MIC) ranging from 0.0063 to 0.2 g/ml. However, it was inactive for Gram-negative bacteria and animal assays. As more evidence is obtained, it is becoming abundantly clear that bacteria form highly specific, symbiotic relationships with marine plants and animals. Experience in this area arose from a study of the pathogen resistance of the estuarine shrimp Palaemon macrodactylus. Gil-Turnes et al., (1989) observed that the eggs of P. dactylus possess significant bacterial epibionts, which, when removed by treatment with antibiotics, leads to the rapid infestation of the eggs by pathogenic fungi, especially of Lagenidium callinectes. It could be due to the anti-fungal agents produced by bacteria. Studies on marine bacterial products in Mariculture are therefore essential.

Marine fungi

Although terrestrial fungi have represented a major biomedicinal resource (penicillin from Penicillium, for example), studies to develop the biomedicinal potential of marine fungi were less The isolation of a small lactone, leptosphaerin from Leptosphaeria oraemaris by Schiehser (1980) demonstrated that marine fungi may form important resource for unique metabolites. Later, the useful chemical, Gliovictin was isolated from marine fungus, Asteromyces cruciatus (Shin and Fenical, 1987). Since then more than twenty useful bioactive compounds have been derived from marine fungi.

Marine Microalgae

Microalgae are significant resource for bioactive metabolites, particularly cytotoxic agents with applications in cancer chemotherapy (Moore et al., 1988). From the marine microalgae such as from the blooms of Phaeocystis sp., antibiotic substances were listed. Phaeocystis pouchetii is reported to produce chemicals such as Acrylic acid, which constitutes about 7.0% of the dry weight. The antibiotic substances thus produced are transferred throughout the food chain and found in the digestive tract of Antartic penguins (Sieburth, 1961). Production of ß carotene and vitamins by the halotolerant alga Dunaliella sp., is documented. These compounds have much importance for the Mariculture activities.

Marine Macroalgae

Of the total marine algae so far evaluated, about 25.0% showed one or the other biological activity. The metabolites of green algae were reported to contain 1,4 –diacetoxic butadiene moiety, which exhibited icthyotoxic property. Among the red algae, halogenated lipids have been isolated, particularly from the Laurencia sp. The rare chemical prostaglandin was also reported to occur in Gracilaria pichenoids. Ulva meal supplementation was found to provide disease resistance to red sea bream in Japan (Satoh et al, 1986). Similar results were also reported from Japan on the use of Ulva meal supplementation towards disease resistance and high growth rate in black sea bream (Nakagawa et al, 1987). The polysaccharide fractions from marine algae, Porphyra yezoensis (PASF) was found to stimulate the in vivo and in vitro murine phagocytic function. The purified fractions of PASF gave stronger phagocytic activity (Yoshizawa et al. 1995). Some of the macro algal crude extracts indicated their potential therapeutic nature when challenged with potential pathogens among fish and shellfish. The cellular and humoral responses of the fish/shellfish towards the algal metabolites were investigated (Lipton, 2001).

Marine Sponges

The wider biosynthetic capability of sponges could be attributed to their biological association with other symbionts. According to Bertrand and Vacelet (1971), about 38% of the sponge body comprises of microorganisms. A wide variety of secondary metabolites were isolated from sponges and these have been associated with antibacterial, antimicrobial, antiviral, antifouling, HIV-protease inhibitory, HIV reverse transcriptase inhibitory, immuno- suppressent and cytotoxic activities. In addition to potential anticancer applications, the MNPs of sponges have a myriad of activities ranging from antibiotic activity including anticoagulant, antithrombin, anti-inflammatory, as well as imunomodulatory activities.

Presence of specific symbiont morphologies of bacteria within specific sponges has been reported. These specific bacteria, which live symbiotically with sponges, passed through their feeding chambers without being digested. This suggested some sort of encapsulation or recognition process. In the demosponge, Halichondria panicea, an association with the microbe Pseudomonas insolita was suggested to be lectin-based (Müller et al., 1981). Wilkinson found an immunological basis for symbiosis in some sponges, which he claimed as evidence of a Precambrian origin for many symbioses (Wilkinson, 1984).

A major problem with the early studies on sponge-microbe symbiosis was that most microorganisms were uncultured or unculturable. The application of molecular biology to spongemicrobe symbiosis is now yielding results that could not have been obtained by classical microbiological methods. The discovery of a member of the Archaea living specifically within a sponge similar to Axinella mexicana was particularly worth to mention (Preston et al., 1997).

Avenues for further research and development in relevance to Mariculture:

Perusal of literature indicates that during the last three decades number of diverse biologically active compounds has been isolated from marine organisms, but the number of compounds taken-up for the field trial/clinical use are scanty. Some of the future requirements are listed below:

1. Microbial Isolation/screening and culture techniques: required as new symbiotic microbes are difficult to culture under laboratory conditions. Basic Research in Marine Microbiology is essential. Without considerable attention to developing the basic biology of marine microorganisms, explorations for new bioactive metabolites would be limited to those few classes of microorganisms, which are readily isolated and grown under "standard" conditions. Unfortunately, little is known about the specific nutrients and growth factors required by most of the marine microbes. For example, the common media components such as peptone, sugars etc., are unrealistic marine nutrients as complex carbon sources such as chitin, sulphated polysaccharides, marine protein etc., are found in the marine habitat. In addition, information is lacking on some of the uncommon inorganic elements such as lithium, silicon etc., abundant in the marine sediments. As a result of these difficulties, it is seen that less than 5% of the available microbial population is only culturable under the standard laboratory conditions. Presently, this condition, certainly limits the scope and ability to isolate and culture majority of the interesting and new microbes.

2. Preparation of crude extracts for bioactivity: as the goal is to obtain the widest possible screening for each crude extract so that no useful compound is over looked. Solvents such as methanol, chloroform or ether as independent solvents or as combinations can be used depending upon the nature of the MNPs. As soon as the crude extracts are obtained, there is need for immediate and simple in vitro assays such as: i. Antimicrobial and ii. Enzyme inhibition assay (very low quantity of sample only is required). This in turn helps in the `bioassay – guided fractionation and purification’ process.

3. Purifucation: Once bioactivity is detected in the crude extract, the next step is to purify the same. It is important to employ non-destructive method such as spectroscopic method, which conserves the materials for further bioassays. In addition, techniques such as: TLC, MS/IR/uv and H nMR – (for structural elucidations) are to be adopted for purification of the crude extract and for determining the structure.

4. Pharmacological screening: The next step after purification and structural elucidation is pharmacological screening. Studies such as determining the LD50 of the extracts in mice, in addition to brine shrimp assay, fertilized sea urchin assay and starfish assay are to be carried out in established laboratories. Further tests such as: antiviral (AIDS/anti- HIV), cytotoxic, anti-inflammatory, anti-tumor, tumor promoter (protein kinase), analgesic, anti-coagulant / anti-thrombic (ex: heparin), anti-ulcer, anti-cholesterol / anti-lipemic, wound dressing, anti-parasitic, anti-protozoa are to be conducted.

5. Commercial development of bioactive (MNP) products: The `co-operative drug development programme’ as suggested by Dr. Faulkner is the best method, which will solve the problems arising on issues such as: patent rights, academic freedom and industrial secrecy.

6. Conservational aspects of source organisms: Eco-friendly collection of the source organism and required supply of them in bulk for scaling-up process.

7. The role of Industry and Academia: Considering the less microbiological and intensive pharmacological training to the industrial personnel, relevant microbiological training has to be imparted to the industrial pharmacologists. New isolation methods, media development etc., are to be included in the curricula of academic/research institutes. Collaborative programmes which combine biomedical and microbiological expertise of the pharmaceutical industry with the marine microbiological resources available in the marine R&D Institutes will in the long run help in the better utilization of the marine resources for biotechnological aspects.