Project: Alternative antifungal agents from natural sources for aquaculture and test methods for ecotoxicity and antifungal effects

The project will be performed in 3-stages:_x000D_o 1st Semantic modelling of the literature and screening of antifungal agents from natural sources. An antifungal screening test system will be developed._x000D_o 2nd Structure analysis of the effective components and characterization of their effects on waterborne pathogenic fungi. Environmental safety will be tested as antibacterial effects on environmental bacteria, a system to be developed._x000D_o 3rd Patent application, (clinical trial) and commercialisation of the compounds and of the testing systems developed._x000D__x000D_Background _x000D_Malachite green (MG), a triarylmethane type of dye, is an extensively used biocide in the aquaculture industry worldwide. It is highly effective against important protozoal and fungal infections._x000D__x000D_However, despite its extensive use as an antifungal agent MG is a highly controversial compound due to its toxic properties, such as carcinogenesis, mutagenesis, teratogenesis and toxicity to the respiratory tract. The use of this dye is restricted or prohibited in several countries: In 1981, the United States prohibited the use of the substance in food-related applications. EU and Norway placed a similar ban in 2002. Japan in July 2003 revised its Pharmaceutical Affairs Law, which limited the use of malachite green._x000D__x000D_Alternative chemical agents such as formaldehyde, hydrogen peroxide potassium permanganate, polyvidone iodine, copper sulphate and isopropyl alcohol have been explored but proved unprofitable. Besides, there are safety concerns, because of suspected carcinogenicity and potential adverse effects on the environment. _x000D__x000D_Working plan_x000D_1) Screening of various natural antifungal agents and determination of their effects_x000D_Candidate plants will be screened by a bio-informatics system, semantic modelling, in which they will be selected from literature databases. In order to do antifungal in vitro tests, fish borne pathogenic fungi have been isolated from fish farms. A rapid and simple antifungal test system will be developed._x000D__x000D_2) Structure analysis and safety evaluation of effective compounds._x000D_After the pre-screening of toxicity by the Brine Shrimp assay (Meyer et al. 1982), physical and chemical structure will be identified by IR, Mass spectrophotometer, Polarimeter, TLC and UV spectra analysis. _x000D_Haemolytic activities on human and fish erythrocytes and intraperitoneal mouse LD50 will be measured._x000D_In order to screen for ecotoxicity, a rapid and simple antibacterial test system will be developed, based upon the MARA technology on selected marine bacteria._x000D__x000D_3) Toxicity/Safety and effectiveness of product_x000D_This will be assessed through standard methods:_x000D_- Tolerability, side effect, physiological function and metabolism of antifungal agent in fish and determination of toxicity to mammals (mice) (Phase I)._x000D_- Optimum dose calibration by determination of side effects, hazards, and expected indications through short-term administration to fish (Phase II)._x000D_- Dose effect of antifungal agent will be determined in vivo in infected trout, eel and their fertilized eggs with water borne fungi from fish farms. _x000D_- Effectiveness, safety, availability will be determined and followed by an open and comparative clinical trial (Phase III)._x000D__x000D_4) Commercialisation_x000D_Investigations by applicable pharmaceutical industry:_x000D_- Decrease in the purification time._x000D_- Increase in the body availability._x000D_- Optimization of the extraction methods suitable for the effective compounds._x000D_- Improvement of the environmental stability. _x000D_- Transfer of developed technologies into suitable test kits for commercial purposes of the antifungal screening test and of the ecotoxicity test._x000D_- Patent application for the searching methods for antifungal compounds from natural material._x000D_- Improved methods for extraction, isolation and analysis of antifungal agents and effective compounds in natural material and patent these to a great extent._x000D_- Construct the database (chemical structure, property, toxic effects etc.,) of antifungal agents._x000D__x000D_Commercial products_x000D_o A natural antifungal agent that is effective in aquaculture but has little effect on the environment._x000D_o A test kit for screening of antifungal activities._x000D_o A test kit system for screening of ecotoxicity based upon the MARA system._x000D__x000D_§. MARA (Microbial Assay for Risk Assessment) system_x000D_The test is a patented method for measurement of toxicity developed upon the activities against an array of at least 11 microorganisms prepared in a microplate. After incubation, all results are obtained and calculated by the MARA computer software. (For additional information -

Acronym MALGA (Reference Number: 4362)
Duration 01/05/2008 - 30/04/2011
Project Topic The aim of this project is to find natural antifungal compounds as less toxic alternatives to Malachite Green to be used in the aquaculture industry and to develop suitable test kits for screening and environmental safety evaluations.
Network Eurostars
Call Eurostars Cut-Off 1

Project partner

Number Name Role Country
3 PhPlate Microplate Techniques AB Coordinator Sweden
3 Microbial. Sistemes i Aplicacions Analítiques SL Partner Spain
3 BINEX Co., Ltd. Partner Korea, Republic of