Tez özetleri Astronomi ve Uzay Bilimleri Anabilim Dalı 2

Development of a Novel Spectrofluorometric Method for the Hypochlorous Acid Scavenging Activity Measurement of Polyphenols
Hypochlorous acid (HOCl) is an important reactive oxygen species (ROS), non-radical, and taking part in physiological processes concerned with the defense of the organism. When the production of HOCl is not sufficiently controlled, this may give rise to adverse effects (i.e., inflammatory diseases, atherosclerosis, chronic inflammation and some cancers) in the organism parallel to a weakening of the antioxidant defense, similar to other ROS. Polyphenolic compounds as scavengers of HOCl, could have potential therapeutic effects in diseases in which this oxidant (a highly reactive oxidant produced by activated phagocytes) plays a pathogenic role. Flavonoids, simple phenolic acids are polyphenolic substances present in food plants and have been extensively studied for their scavenging acitivities various free radicals (i.e., hydroxyl radicals, superoxide, nitric oxide, and peroxyl radicals), but there has been limited information regarding its scavenging by polyphenolic compounds. Generally, although there are quite a number of methods in literature for the determination of HOCl scavenging activity, all these methods suffer from certain restrictions. For all these reasons, it is evaluated that novel methods for the measurement of HOCl scavenging activity applicable to polyphenolic compounds and their real mixtures need to be devised to eliminate the aforementioned restrictions and to fill in this literature gap. In this regard, a new fluorometric HOCl scavenging activity assay was developed in this thesis. Resorcinol (1,3-dihydroxybenzene) was selected as the probe molecule for the developed method. Although it has been reported in literature that resorcinol is a fluorescent molecule that can be chlorinated with HOCl, it is the first time in this thesis for resorcinol to be used as a probe for HOCl scavenging activity determination of polyphenolic compounds. The fluorescence intensity of resorcinol is attenuated as a result of its reaction with HOCl. Resorcinol is fluorescent, but its chlorination products are not. The attenuated intensity of resorcinol fluorescence is dependent upon the HOCl scavenging activity of the tested compound as measured by competitive reaction kinetics equation. With the aid of resorcinol fluorescence values recorded in the presence and absence of phenolic scavengers, the IC₂₅ (25% inhibitive concentration) of phenolics (polyphenols: flavonoids, simple hydroxybenzoic and hydroxycinnamic acids, etc.) and real samples (some herbal teas) were calculated. The scavenging activities of polyphenolic compounds tested with respect to the developed method decrease in the order: epigallocatechin gallate > quercetin > gallic acid > rutin > catechin > kaempferol. The present study revealed that epigallocatechin gallate (IC₂₅=0.1 µM) was the most effective scavenger agent. In the preparation of herbal tea samples for analysis, the method of microwave extraction was used, having distinct advantages of reduced cost and time and increased reproducibility over classical extraction methods (such as soxhlet extraction, liquid-liquid extraction, column chromatography and preconcentration techniques). The results obtained with the developed method for polyphenolic compounds and herbal teas were statistically compared (using the ANOVA test) to those found by the reference methods KI/taurin and HPLC. The obtained results demonstrate that green tea extract exhibited the highest HOCl scavenging activity (Inhibition, %) by a large difference in both assays (developed method and KI/taurin reference method) (54.8% and 30.4%, respectively).
In conclusion, a fluorometric method for more sensitive, simple and selective measurement of HOCl scavenging activity of antioxidants (individually and in complex matrices) compared to the existing methods in literature was developed. The basic advantages of such a method can be summarized as follows: the method is not enzyme−based, prone to relatively easy inhibition; the optimized probe is selective toward the HOCl analyte (i.e. not reacting with other possible interferents in the medium); the analytical wavelengths of excitation and emission (λ_ex=277 nm, λ_em= 304 nm) are basically characteristic for the probe (resorcinol); the method is more sensitive than absorptimetric methods; the method is less costly and laborious than the corresponding HPLC methods.