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Identifier uch.med.phd//2000tzatzarakis
Title Σύνθεση και βιολογική έρευνα χημειοθεραπευτικών ελεγχόμενης απόδοσης για τον έλεγχο των μυκοτοξικώσεων και μυκητιάσεων
Alternative Title Synthesis and study of the biological activity of new controled release chemotherautic agents for the prevention of mycotoxicosis and the treatment of fungal infections
Creator Tzatzarakis, Manolis
Abstract The present dissertation deals with the problem of mycotoxicoses, the presence of mycotoxins in food, methods of mycotoxin detection and methods of protection of products by the synthesis of new biocompatible polymeric systems that inhibit fungal growth and mycotoxins formation. The dissertation can be divided into three parts. In the first part, the methods for the qualitative and quantitative determination of aflatoxins B1, B2, G1, G2, M1 and M2 are described. The techniques utilized were thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) A mixture comprising of chloroform-acetone (9:1) was used as optimal for the development of aflatoxins on the TLC plate. The detection limit of this method for aflatoxin B1 was 8 ng, for B2, G1 and M2 was 10 ng while for G2 and M1 was 15-20 ng. The conditions used for HPLC analysis of aflatoxins were as follows: A C18 column was used and the mobile phase comprised of a water-acetonitrile-methanol (50:30:20v/v) mixture. The aflatoxins were detected by a UV detector at 365 nm. The retention time of aflatoxin B1 was 10.30 min, of aflatoxin B2 was 8.93 min, of aflatoxin G1 was 8.01 min and of aflatoxin G2 was 7.21 min. The detection limit of aflatoxin B1, B2, G1 and G2 was 50 ng/ml, 150 ng/ml, 100 ng/ml and 250 ng/ml respectively. Two different methods (method A and method B) were used for the detection and quantitative determination of aflatoxins from food (corn, soy, and mixtures of food with corn or soy as the main ingredient). TLC in method A was used in order to determine aflatoxins qualitatively and to isolate them from food componets. The quantitative measurement of aflatoxins was performed by high-pressure liquid chromatography. The detection limits of aflatoxins in both of the methods were 4-5 ng/g for aflatoxins B1 and G1, 6 and 8 ng/g for the aflatoxins B2 and G2. The mean recovery of method A was 62.17 % and of method B was 73.39%. In the second part, the synthesis of novel, water-soluble polymeric forms of sorbic acid, nystatin and amphotericine B as well as the study of their physicochemical properties are described. The new products are controlled release substances with mycostatic and antifungal properties-activity. These polymeric forms of sorbic acid, nystatin and amphotericine were prepared in order to confirm their biological activity and study their physicochemical properties, to reduce their toxicity and to improve their bio-compatibility. Polymeric esters of sorbic acid with molecular weight 10*103, 40*103 and 360*103. were prepared Sorbic acid was connected to the polymeric carrier of polyvinylpyrrolidone (PVP) with a covalent bond. The mean % content of sorbic acid in these polymeric esters ranged between 3.4 and 5.1 %. Complexes of sorbic acid with molecular weight 10*103, 25*103, 30*103, 40*103, 90*103 και 360*103 were prepared using two different methods. In the complexes, sorbic acid is attached on PVP with hydrogen bond. In the first method, sorbic acid reacted with PVP in water and its content ranged between 2.5 mass % to 5.5 mass %. In the second method, sorbic acid reacted with PVP in an organic solvent like ethanol, methanol, isopropanol, dimethylformamide, etc and the maximum content of sorbic acid was 50 mass %. Nystatin and amphotericine B complexes were prepared using similar methods. The maximum content of nystatin in its complexes was 0.25 to 0.5 mass % while the content of amphotericine in its complexes was 0.167 to 0.333 mass %. The molecular weights of complexes of nystatin and amphotericine were 10*103, 25*103, 30*103, 40*103, 90*103 και 360*103. The structure and physicochemical properties of the resulting sorbic acid complexes were investigated by IR spectroscopy, viscosity and permeability studies. Our aim was to determine the effect of molecular weight of PVP in the synthesis and stability of complexes, the effect of the organic solvent (ethanol, methanol, isopropanol, dimethylformamide etc) in the synthesis of complexes, the kind of chemical bond between sorbic acid and PVP, the differences in the physicochemical properties of PVP and sorbic acid after the synthesis of complexes. We observed that the reaction of sorbic acid and PVP was easier when low molecular weight PVP was used. Also, the formation of complexes in organic solvents in which PVP gave high values of specific viscosity was more successful. It was observed that the value of specific viscosity in the produced sorbic acid complexes remained constant with increasing concentration, something that was associated with the formation of hydrogen bonds and confirmed the interaction between sorbic acid and PVP. IR spectra indicated that the carbon chain of sorbic acid was linear, without any circular dimers, while the π-electrons of the carbonyl group were in conjunction with the π-electrons of the acid double bonds. The presence of free acid in the complexes decreases with increasing complex molecular weight. This implies that the formation of the complexes depends on the molecular weight of PVP. The amount of water present in the crystalline structure of PVP is very important. It was found that the presence of free acid in the complexes increases proportionally to the amount of water in PVC. The interaction of PVP and sorbic acid by hydrogen bonding is detected by absorbance at 2400-2600 cm-1 which is characteristic of this type of bond. In the third part of the dissertation, we compared the mycostatic activity of common food additives against the non-pathogenic fungus Fusarium oxysporum radicis- cucumerinum and the pathogenic fungi Aspergillus parasiticus, Penicillium viridicatum, Candida albicans and Candida tropicalis. The additives studied were sorbic acid, potassium sorbate, propionic acid, acetic acid, formic acid and sec-butylamine. The above organic acids possess antifungal and antibacterial properties. Recent studies have shown that sorbic acid and propionic acid reduce aflatoxins (mycotoxins) biosynthesis as well. Our results proved that the most effective growth inhibitor of Fusarium oxysporum cucumerinum was sorbic acid, followed by potassium sorbate, propionic acid, acetic acid, sec-butylamine and formic acid. The activity of organic acids increased with increasing number of carbon atoms and molecular weight. The effectiveness of sorbic acid was approximately 6.7 times higher than that of formic acid, 4 times that of acetic acid and sec-butylamine and 2.1 that of propionic acid and slightly higher than potassium sorbate. Although sorbic acid is a very effective mycostatic agent with low toxicity to human and animal, its use is limited due to its low solubility in water. Similar problems appear in the use of amphotericine B, nystatin and many azoles with low water solubility. The complexes of sorbic acid proved to be more effective than the polymeric esters of sorbic acid. Their effectiveness increased with decreasing molecular weight. Complex 1 with molecular weight 10*103 was the most effective growth inhibitor against Fusarium oxysporum when compared to sorbic acid, potassium sorbate and the rest mycostatic agents that were examined. Nystatin and amphotericine B belong to the family of polyenes and clotrimazole, econazole and miconazole belong to the family of azoles. The above substances posses antifungal properies and are used in the therapy of fungal infections. Azoles are more effective growth inhibitors than polyenes against Fusarium oxysporum. The inhibitory efficacy of these chemicals decreased in the following order: econazole, clotrimazole, miconazole, amphotericine B and nystatin. Nystatin is less effective than the other antifungals but it is the only antifungal agent used in veterinary medicine in our country. Nystatin was a more effective inhibitor than amphotericine in the spore germination of Fusarium oxysporum, while the azole mycostatic agents were no effective at all. The complexes of nystatin were more effective than nystatin in aqueous solutions against the growth and spore germination of Fusarium oxysporum. Their activity depended on their molecular weight. Particularly, complex 6 with molecular weight 360*103 is 25.36 times more effective than nystatin. It is followed by complex 4 with molecular weight 40*103 and the complex 1 with molecular weight 10*103. The high effectiveness of sorbic acid, nystatin and amphotericine complexes was confirmed against pathogenic species like Aspergillus parasiticus, Penicillium viridicatum, Candida albicans και Candida tropicalis.The complexes 1, 2 and 3 show higher activity against fungi than sorbic acid. Complex 1 of sorbic acid was 3 times more effective than sorbic acid and 4 times more effective than potassium sorbate against Aspergillus parasiticus. Also it was 2.5 and 3.75 times more effective than sorbic acid and potassium sorbate against Penicillium viridicatum. It was also more effective against Candida albicans and Candida tropicalis. Complex 1 of sorbic acid proved to be fungicidal against Candida albicans in contrast to sorbic acid and the other complexes with higher molecular weight, which were fungistatic. The activity of all sorbic acid complexes depends on the structure and it increases with decreasing molecular weight. Complexes 1, 2 and 3 of amphotericine showed to have similar activity with amphotericine while complexes 4, 5, 6 were less effective against the pathogenic species examined. Nystatin complexes act in a similar way to complexes of amphotericine but are slightly less effective. The MICs values of these complexes decreased with increasing their molecular weight confirming the relationship between biological activity and their structure. We can summarize the advantages of sorbic acid, nystatin and amphotericine complexes as follows: They are pure substances, free from contaminants, with stable structure, relatively simple, fast and cheap production process, they are highly soluble in aqueous solutions, their activity can be controlled via their structure, they are highly biocompatible and more effective compared to the parent compounds. The polymeric complexes of sorbic acid may be used either as food additives for the prevention of microorganism infection or as chemotherapeutic agents. The polymeric complexes of nystatin and amphotericine B may either be used for the prevention of gastrointestinal tract colonization by Candida or other possibly pathogenic fungi in patients susceptible to fungal infection (e.g. immunosuppressed, cancer patients) or in cases of infection by hyphomycetes like Aspergillus and Penicillium.
Issue date 2000-07-01
Date available 2000-09-26
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/d/3/d/metadata-dlib-2000tzatzarakis.tkl Bookmark and Share
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