Purification and characterization of secondary metabolites of picoa juniperi, picoa lefebvrei and investigation of their biological activities

In this thesis, the biological activities of Picoa juniperi and Picoa lefebvrei species which are naturally distributed in Anatolia and have commercial potential, were investigated. In this study, Picoa juniperi Vittad collected from Elazığ-Malatya and Picoa lefebvrei (Pat.) Maire collected from Denizli and their species were dried under appropriate conditions and extracted with n-hexane, chloroform, acetone and methanol respectively under room conditions. Soaked in methanol extract, the residues of truffle were extracted with water at 80˚C and 5 different extracts of different polarities were obtained. To determine the biological activities, antioxidant activities and enzyme inhibition activities (anticholinesterase and urease) of each extract obtained from these species, as well as their toxicity against Lung Cancer (H1299), Breast Cancer (MCF-7), Prostate Cancer (PC-3) cell lines and Mouse Fibroblast cells (L929) were tested in vitro conditions. Antioxidant activity tests of the extracts were investigated with 5 different methods that are β-carotene bleaching method, DPPH free radical scavenging activity, ABTS cation radical scavenging activity, CUPRAC method and metal chelating capacity respectively. Anticholinesterase enzyme activity was determined by using acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzyme inhibition activity methods. 800 µg/mL DPPH free radical reducing activity inhibition of P. juniperi and P. lefebvrei's methanol extracts are 58.27±0.19% and 57.54±0.01% respectively; while their ABTS cation radical reducing activity inhibitions are calculated as 91.56±0.10% and is 91.47±0.02%. The urease enzyme inhibition activities of methanol and water extracts of both truffle species showed moderate activity compared to thiourea which sets a standard (53.79±0.40%, 40.96±0.07%; 55.42±0.86%, 41.59±0.74%). Toxicities of the extracts against the abovementioned cancer cells were determined by calculating their 50% viability. According to these results, water extract of Picoa lefebvrei is highly toxic to Lung Cancer (H1299) and Prostate Cancer (PC-3) cell lines, while its toxicity on L929 fibroblast cell is low. On Prostate Cancer (PC-3) cell line, P. lefebvrei methanol extract decreased to 38.2% while P. juniperi methanol extract decreased to 49.9% viability. On breast cancer cell line (MCF-7), P. lefebvrei methanol extract decreased to 41.5%, whereas Picoa juniperi decreased to 54.2%. In the study methanol extracts of both species had high toxicity on Breast Cancer (MCF-7) and Prostate Cancer (PC-3) cell lines, however, P. lefebvrei water extract was much more effective on Lung Cancer (H1299) cell line than the other extracts (25.6% at 200 µg/mL). According to the antioxidant and cytotoxic activity results, methanol and chloroform extracts of both species were isolated while their activities are controlled. In this respect, the isolation of bioactive compounds obtained from n-hexane, chloroform, acetone, methanol and hot water extracts was carried out by using column chromatography, thin-layer chromatography and preparative HPLC techniques. The structures of the purified compounds were elucidated by 1H-NMR, 13C-NMR, 2D-NMR, FTIR and MS spectra techniques. Structures of 17 compounds obtained from Picoa lefebvrei and Picoa juniperi were fully elucidated. The compounds obtained from Picoa juniperi are Brassıcasterol (1), Ergosterol (2), Ergosterol endoperoxide (3) Ergosta 5,22-diene 3-O-β-ᴅ-glucopyranoside (4), Juniperon A (5), Juniperon B (6), Mannitol (7), Adenosine (8), Linoleic acid, Oleic acid (9). The compounds obtained from P. lefebvrei are as follows: Brassıcasterol (10), 5,22-diene 3-O-β-ᴅ-glucopyranoside (11), Uracil (12), 1-β-L- xylofuranosyl (13), Erythriol (14), α-ᴅ-glukopiranozil-α-ᴅ-glukopiranozit (15), Brassicasteryl linoleate (16); 5α-6α epoxy ergosta-7,22-dien-3β-ol (17). Juniperon A (5) and Juniperon B (6) were also isolated and their structures were elucidated for the first time by this thesis. Moreover, polysaccharides of both species were removed by appropriate methods and their structures were characterized by HPLC and FTIR. Antioxidant activities of purified polysaccharides were identified. Besides, phenolic compounds of both species were investigated by HPLC-DAD system. According to the results, fumaric acid is the main compound in both P. juniperi and P. lefebvrei species (165.2 µg/g; 12.6 µg/g). In this study, DPPH free radical scavenging, ABTS cation radical scavenging of the pure compounds with sufficient amount out of 17 isolated compounds, as well as their toxicity against Breast Cancer (MCF-7), Mouse Fibroblast cells (L929) were identified in vitro tests. Among other compounds whose structures were elucidated, it is identified that Brassicasterol, Ergosterol, Ergosta 5,22-diene 3-O-β-ᴅ-glucopyranoside, Adenosine, 1-β-L- xylofuranosyl, 5α-6α epoxy ergosta-7, 22-dien-3β-ol have toxicity against cancer cells, while 1-β-L- xylofuranosyl, Adenosine compounds were found to have antioxidant effects.