Study On Synthesis And Biological Activity Of Phloretin Acyl Hydrazones

Phloretin, which is an active substance, a kind of dihydrochalcone, abundantly present in pericarp and velamen of apples, pears, berries and other fruits. At present, phloretin is used as a new type of cosmetic additives in foreign countries, because phloretin may dilute the melanin, whitening skin and the effect is better than the present commonly used kojic acid and arbutin in cosmetics. In addition, phloretin posses lots of biological activities. Antioxidant, hpyerglycemic, antitumor, anti-inflammatory has been reported. Currently, the research on phloretin is mainly focused on the biological activity; the field on the chemical modification is still not fully open up. Tyrosinase is a copper-containing multifunctional oxidase, is known to be a key enzyme for biosynthesis of melanin in organism. Tyrosinase inhibitors have attracted much attention recently. In this paper we choose phloretin as a ligand, and synthesised three novel compounds with benzoyl hydrazine, salicylyl hydrazine and4-hydroxybenzhydrazide. The three Acyl hydrazone derivatives were characterised by UV, IR, NMR, MS spectrum. We hope to improve the biological activity of phloretin through chemical modification and provide a theoretical basis to the structure research of phloretin. This thesis consists of four parts:Chapter one, reviewed the international research status of phloretin, and then took a brief inroduction of biological function of hydrazone compounds, then reviewed the international research status of tyrosinase and tyrosinase inhibitor, clarify the direction and significance of this research.Chapter two, selected benzoyl hydrazine, salicylyl hydrazine and4-hydroxybenzhydrazide, a series of phloretin hydrazone derivatives were synthesized:phloretin benzoyl hydrazone(1), phloretin2-hydroxy benzoyl hydrazone (1) and phloretin4-hydroxy benzoyl hydrazone (3), the three derivatives were characterised by UV, IR, NMR, MS spectrum.Chapter three, The antioxidant experiment including reduction of Fe3+ability, scavenging DPPH radical and ABTS radical ability and protect protein oxidative damage ability. The results showed that compounds exhibit different activities in different experimental models. The three new compounds shows better antioxidant activities than phloretin. The structure-activity relationships were analyzed. The compound2and3shows higher antioxidant activities than compound1, with its aroyl containing hydroxyl group in the ortho-position and para-position, respectively, which showed that the number and the location of hydroxyl group in the aroyl benzene ring were assumed to play key roles in the antioxidant activities of the compounds.Chapter four, the tyrosinase inhibition activity of phloretin, compound1-3were investigated, which were compared to their matrix phloretin and the result is satisfactory. Compound2(IC50=13.4×1.1μM) and compound3(IC50=19.1±0.82μM) show higher inhibition activity than Phloretin (IC50=23.7±1.8μM). The tyrosinase inhibitory activity is not only dependent on the number but also the location of hydroxyl group in the compounds. The enzyme inhibition kinetics was study. The inhibition mechanisms of four compounds against the enzyme were reversible. The inhibition type of inhibitions on tyrosinase was mix-type. Compounds inhibit enzyme activity is by lowering the enzyme activity, and not reduce the effective quantity of enzyme. The underlying tyrosinase activity inhibition mechanism was investigated by fluorescence spectra. With an increasing concentration of compound2, the fluorescence intensities of tyrosinase were decreased, more interestingly, a new peak appeared and upon the accumulation of compound it was increased obviously. The results indicated that the quenching of tyrosinase by phloretin and compound1,3was static quenching owing to1:1complex formation. The compound-Cu2+interaction were studied by absorption spectroscopy. The presence of Cu2+resulted in spectral change of compound1-3. These differences in the tyrosinase inhibitory activity for the hydrazones were related to its aroyl containing hydroxyl group and the ability to form the chelate with copper ions.