Optimization of the technological procedure for isolation of amygdalin and quercetin from plants and their pharmacological activity

The plant extracts and compounds isolated from natural sources are commonly used for prevention of chronic diseases. These bioactive compounds have an increased application in modern conventional medicine. Despite a great progress in the treatment of malignant tumors, this disease still takes many lives. Today, a huge number of chemotherapeutics for tumor treatment can be found in the market. Because of their toxicity most drugs and drug products can not be applicated in the doses that completely inhibit the cells growth in organism. Quercetin and amygdalin belong to the most commonly used pharmacological agents for tumor treatment. Therefore, the subject of this dissertation is isolation of amygdalin and quercetin from two plant materials, i.e. green tea leaves and plum seeds. In this study, the effect of extraction time, solvent nature and solid to liquid ratio was investigated for quercetin, i.e. total flavonoids from green tea leaves using experimental design and artificial neural network. In addition to these parameters, the impact of extraction temperature was also studied during amygdalin extraction from plum seeds. The content of amygdalin, as well as quercetin in the extracts was determined using the developed HPLC methods. Total flavonoids content was determined by a spectrophotometric method based on the formation of complex with aluminium chloride. Amygdalin was isolated from the ethanolic extract of plum seeds after addition of diethyl-ether. The obtained amygdalin purity of 90% was defined with regard to the available amygdalin standard using HPLC method. The structural characterization of isolated amygdalin was carried using UV, IR and ESI-MS/MS methods. Adequate methods for determination of antioxidant activity (DPPH assay), antimicrobial activity (disc diffusion method) and antiproliferative activity (MTT assay) were used in order to define the pharmacological activities of green tea and plum seeds extracts, isolated amygdalin, quercetin and amygdalin standards. Based on all these pharmacological studies, it was shown that quercetin and green tea extract have better antioxidant activity compared to amygdalin and plum seeds extract. Amygdalin standard and plum seeds extract have antibacterial activity only on the strain of P. аeruginosa (>23 mm for both samples) and E. coli (17-19 mm and 20-22 mm, respectively). Abstract Quercetin standard and green tea extract have an additional effect on the strain of S. aureus with an inhibition zone of 20-22 mm and 17-19 mm, respectively. IC50 values of isolated amygdalin and its standard (>379.5 μg cm-3 and 304.65-333.27 μg cm-3, respectively) for the investigated cell lines were higher compared to green tea extract and quercetin standard (47.41-114.39 μg cm-3 and 19.24-44.07 μg cm-3, respectively). These values were determined using MTT assay and are in accordance with the literature. The cytotoxicity of amygdalin can be increased by addition of β-D-glucosidase enzyme