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Hypoxia is a crucial factor in cancer therapy, determining prognosis and the effectiveness of treatment. Although efforts are being made to develop methods for assessing tumor hypoxia, no markers of hypoxia are currently used in routine clinical practice. Recently, we showed that the combined endogenous MR biomarkers, R and R *, which are sensitive to [dissolved O ] and [dHb], respectively, were able to detect changes in tumor oxygenation induced by a hyperoxic breathing challenge. In this study, we further validated the ability of the combined MR biomarkers to assess the change in tumor oxygenation induced by an allosteric effector of hemoglobin, myo-inositol trispyrophosphate (ITPP), on rat tumor models. ITPP induced an increase in tumor pO , as observed using L-band electron paramagnetic resonance oximetry, as well as an increase in both R and R * MR parameters. The increase in R indicated an increase in [O ], whereas the increase in R * resulted from an increase in O release from blood, inducing an increase in [dHb]. The impact of ITPP was then evaluated on factors that can influence tumor oxygenation, including tumor perfusion, saturation rate of hemoglobin, blood pH and oxygen consumption rate (OCR). ITPP decreased blood [HbO ] and significantly increased blood acidity, which is also a factor that right-shifts the oxygen dissociation curve. No change in tumor perfusion was observed after ITPP treatment. Interestingly, ITPP decreased OCR in both tumor cell lines. In conclusion, ITPP increased tumor pO via a combined mechanism involving a decrease in OCR and an allosteric effect on hemoglobin that was further enhanced by a decrease in blood pH. MR biomarkers could assess the change in tumor oxygenation induced by ITPP. At the intra-tumoral level, a majority of tumor voxels were responsive to ITPP treatment in both of the models studied.

Tumour hypoxia is a well-established factor of resistance in radiation therapy (RT). Myo-inositol trispyrophosphate (ITPP) is an allosteric effector that reduces the oxygen-binding affinity of haemoglobin and facilitates the release of oxygen by red blood cells. We investigated herein the oxygenation effect of ITPP in six tumour models and its radiosensitizing effect in two of these models. The evolution of tumour pO upon ITPP administration was monitored on six models using 1.2 GHz Electron Paramagnetic Resonance (EPR) oximetry. The effect of ITPP on tumour perfusion was assessed by Hoechst staining and the oxygen consumption rate (OCR) in vitro was measured using 9.5 GHz EPR. The therapeutic effect of ITPP with and without RT was evaluated on rhabdomyosarcoma and 9L-glioma rat models. ITPP enhanced tumour oxygenation in six models. The administration of 2 g/kg ITPP once daily for 2 days led to a tumour reoxygenation for at least 4 days. ITPP reduced the OCR in six cell lines but had no effect on tumour perfusion when tested on 9L-gliomas. ITPP plus RT did not improve the outcome in rhabdomyosarcomas. In 9L-gliomas, some of tumours receiving the combined treatment were cured while other tumours did not benefit from the treatment. ITPP increased oxygenation in six tumour models. A decrease in OCR could contribute to the decrease in tumour hypoxia. The association of RT with ITPP was beneficial for a few 9L-gliomas but was absent in the rhabdomyosarcomas.

Tumour hypoxia is a well-established factor of resistance in radiation therapy (RT). Myo-inositol trispyrophosphate (ITPP) is an allosteric effector that reduces the oxygen-binding affinity of haemoglobin and facilitates the release of oxygen by red blood cells. We investigated herein the oxygenation effect of ITPP in six tumour models and its radiosensitizing effect in two of these models. The evolution of tumour pO upon ITPP administration was monitored on six models using 1.2 GHz Electron Paramagnetic Resonance (EPR) oximetry. The effect of ITPP on tumour perfusion was assessed by Hoechst staining and the oxygen consumption rate (OCR) in vitro was measured using 9.5 GHz EPR. The therapeutic effect of ITPP with and without RT was evaluated on rhabdomyosarcoma and 9L-glioma rat models. ITPP enhanced tumour oxygenation in six models. The administration of 2 g/kg ITPP once daily for 2 days led to a tumour reoxygenation for at least 4 days. ITPP reduced the OCR in six cell lines but had no effect on tumour perfusion when tested on 9L-gliomas. ITPP plus RT did not improve the outcome in rhabdomyosarcomas. In 9L-gliomas, some of tumours receiving the combined treatment were cured while other tumours did not benefit from the treatment. ITPP increased oxygenation in six tumour models. A decrease in OCR could contribute to the decrease in tumour hypoxia. The association of RT with ITPP was beneficial for a few 9L-gliomas but was absent in the rhabdomyosarcomas.

Antimicrobial properties of extracts of underground parts of three Laserpitium L. (Apiaceae) species, namely Laserpitium latifolium L., Laserpitium zernyi Hayek and Laserpitium ochridanum Micevski, were investigated. The investigated species are widely used as functional foods, as spices and for preparations in traditional medicine for treating complaints connected with infection and inflammation. Furthermore, antimicrobial and antibiofilm effects of laserpitine, the most abundant compound in the chloroform extract of Laserpitium latifolium, and guaianolide sesquiterpene lactones, such as, isomontanolide, montanolide and tarolide, principal components of the extracts of Laserpitium zernyi and Laserpitium ochridanum were assessed. The antimicrobial activity was tested using the microdilution method against five pathogenic bacteria and five fungi, as well as in the microplate biofilm assay on two Candida clinical isolates (C. albicans and C. krusei). Among the extracts, Laserpitium latifolium showed the most prominent activity. Isolated metabolites exerted higher effects against fungal than against bacterial strains, isomontanolide being the most active. Interestingly, all constituents showed higher potential on inhibition of biofilm formation than fluconazole, a reference compound. Tested metabolites may be good novel agents with high antifungal and antibacterial potential that might find practical applications in food industry as food preservatives in order to retard the growth of food spoiling microbes, but only after detailed safety assessments.

Predmet doktorske disertacije je analiza sekundarnih metabolita i ispitivanje farmakološke aktivnosti hloroformskih i metanolnih ekstrakata i etarskih ulja tri vrste roda Laserpitium L. (Apiaceae): Laserpitium latifolium L., Laserpitium zernyi Hayek i Laserpitium ochridanum Micevski. Laserpitium latifolium je višegodišnja biljka, koja je široko rasprostranjena na evropskom kontinentu. Laserpitium zernyi je skardo-pindska endemična vrsta, sa prirodnim staništem na prostorima Srbije (Kosovo), Albanije, Makedonije i Grčke. Ranije je smatrana podvrstom Laserpitium siler L., subsp. zernyi (Hayek) Tutin. Laserpitium ochridanum je stenoendemit planine Galičice u Makedoniji. Sekundarni metaboliti vrsta roda Laserpitium do sada su analizirani samo u nekoliko šire distribuiranih predstavnika. Od biljaka koje predstavljaju predmet ove doktorske disertacije, široko rasprostranjena vrsta L. latifolium je do sada samo delimično ispitivana u pogledu hemijskog sastava. Druge dve, endemične biljne vrste - L. zernyi i L. ochridanum, nisu hemijski i farmakološki proučavane. Etarska ulja odabranih vrsta roda Laserpitium izolovana su destilacijom vodenom parom i hemijski okarakterisana metodama GC-FID i GC-MS. U etarskim uljima podzemnih organa sve tri ispitivane vrste roda Laserpitium, α-pinen je predstavljao najzastupljeniju komponentu (44,4%, 33,2% i 31,6% u uljima L. latifolium, L. ochridanum i L. zernyi, redom). U etarskim uljima podzemnih organa L. zernyi i L. ochridanum utvrđen je i značajan sadržaj α-bisabolola (30,9% i 10,3%, redom), a u etarskom ulju podzemnih organa L. ochridanum i hamazulena (14,9%). U etarskim uljima plodova L. latifolium i L. ochridanum utvrđen je visok sadržaj monoterpenskih ugljovodonika: u etarskom ulju L. latifolium α-pinena (44,0%), dok je u etarskom ulju L. ochridanum dominanatan satojak limonen (57,7%), a u oba etarska ulja detektovan je značajan procenat sabinena (26,8% i 14,4%, u etarskim uljima plodova L. latifolium i L. ochridanum, redom). U eta, In the scope of the doctoral dissertation are analysis of secondary metabolites and testing of pharmacological activity of chloroform and methanol extracts and essential oils of three species of the genus Laserpitium L. (Apiaceae): Laserpitium latifolium L., Laserpitium zernyi Hayek and Laserpitium ochridanum Micevski. Laserpitium latifolium is perenial species, widely distributed throughout European continent. Laserpitium zernyi is scardo-pindic endemic species, that is distributed in Serbia (Kosovo), Albania, Former Yugoslav Republic of Macedonia (FYROM) and Greece. Previously, it was treated as a subspecies of colsely related Laserpitium siler L., subsp. zernyi (Hayek) Tutin. Laserpitium ochridanum is endemic species at Galičica Mountain, FYROM. Secondary metabolites of Laserpitium species have been so far analyzed just in several widely distributed plants. Considering investigation of the species that are in the scope of this dissertation, only widely distributed L. latifolium has been partially chemically examined. The other two endemic species - L. zernyi and L. ochridanum have not been either chemically or pharmacologically investigated before. Essential oils of selected Laserpitium species are isolated by a steam distillation and chemically characterised by GC-FID and GC-MS. In the essential oils of the underground parts of three Laserpitium species, the main constituent was α-pinene (44.4%, 33.2% and 31.6% in essential oils of L. latifolium, L. ochridanum and L. zernyi, respectively). In the essential oils of underground parts of L. zernyi and L. ochridanum certain content of α-bisabolol was found (30.9% and 10.3%), and in essential oil of underground parts of L. ochridanum was also detected chamazulene (14.9%). The fruit oils of L. latifolium and L. ochridanum were rich is monoterpene hydrocarbons: essential oil of L. latifolium was rich in α-pinene (44.0%), whilst in essential oil of L. ochridanum dominant constituent was limonene (57.7%), and in both oil

In the search for novel cytotoxic agents, sesquiterpenoids and phenylpropanoids have provided interesting lead compounds. From the chloroform extract of the underground parts of Laserpitium latifolium, the daucane sesquiterpenoids laserpitin and acetyldesoxodehydrolaserpitin, and the phenylpropanoids laserin and latifolon were isolated as the major compounds. Acetyldesoxodehydrolaserpitin is identified for the first time in the genus Laserpitium. Using a MTT and a sulforhodamine B (SRB) assay, the cytotoxic and antiproliferative activity of the extract and compounds laserpitin, acetyldesoxodehydrolaserpitin and laserin was tested in two closely related human breast adenocarcinoma cell lines, ie. the invasive MCF 7/6 and the non-invasive MCF 7/AZ. The IC50 values of the extract were in the range of 184.72 - 397.16 mu g/mL, with the most potent effect observed in the MTT test on the MCF 7/6 line. Among the tested compounds, acetyldesoxodehydrolaserpitin exerted a most potent, concentration-dependent effect (IC50 values of 0.60 and 0.51 mu M in the MCF 7/6 cell line, and 2.29 mu M and 31.87 mu M in the MCF 7/AZ cell line in the MTT and SRB test, respectively). The effect of laserin was more pronounced in the MTT test (IC50 of 4.57 and 2.46 mu M in the MCF 7/6 and MCF 7/AZ, respectively).

Chloroform extracts of the underground parts of two Balkan endemic Laserpitium species, Laserpitium zernyi Hayek and Laserpitium ochridanum Micevski, were chemically investigated. Five unknown guaianolides from the class of slovanolides, of which four were additionally 2 beta-esterified, as well as two lactones, previously identified in other Laserpitium species, were isolated from the L. ochridanum extract. From the L. zernyi extract one slovanolide derivative was isolated for the first time in the genus Laserpitium. In addition, the phenylpropanoid latifolone and six known sesquiterpene lactones, characterised as derivatives of slovanolide and silerolide, were isolated from the extracts of both species. The cytotoxic activities of the total extracts and the isolated compounds were tested using MTT and SRB assays on the two human breast cancer cell lines, MCF 7/6 and MCF 7/AZ. The extracts exerted cytotoxic activities with the IC50 values ranging 65.21-348.25 mu g/mL. The L ochridanum extract was most potent in the MTT test with IC50 values of 65.21 and 66.09 mu g/mL in the MCF 7/AZ and MCF 7/6 cell lines, respectively. The highest cytotoxic activity exerted 2 beta,8 alpha-di-angeloyloxy-10 beta-hydroxy-6 alpha H-guaian-3,(7-11)-dien-12,6-olide, a slovanolide derivative with an additional double bond in lactone ring, on highly invasive MCF 7/6 cell line, with IC50 value 0.7 mu M in both assays tested. Generally, guaianolides with a higher number of ester moieties at the positions 2 beta, 8 alpha, 10 beta or 11 alpha exhibited IC50 values in the micromolar range, while eudesmanolides and guaianolides with a lower number of esters did not induce significant cytotoxicity.

Recently, it was shown that the exposure to the potent hop phytoestrogen 8-prenylnaringenin (8-PN) depends on intestinal bacterial activation of isoxanthohumol (IX), but this occurs in only one-third of tested individuals. As the butyrate-producing Eubacterium limosum can produce 8-PN from IX, a probiotic strategy was applied to investigate whether 8-PN production could be increased in low 8-PN producers, thus balancing phytoestrogen exposure. Using fecal samples from high (Hop +) and low (Hop –) 8-PN–producing individuals, a Hop + and Hop – dynamic intestinal model was developed. In parallel, Hop + and Hop – human microbiota-associated rats were developed, germ-free (GF) rats acting as negative controls. IX and then IX + E. limosum were administered in the intestinal model and to the rats, and changes in 8-PN production and exposure were assessed. After dosing IX, 80% was converted into 8-PN in the Hop + model and highest 8-PN production, plasma concentrations, and urinary and fecal excretion occurred in the Hop + rats. Administration of the bacterium triggered 8-PN production in the GF rats and increased 8-PN production in the Hop – model and Hop – rats. 8-PN excretion was similar in the feces (294.1 ± 132.2 nmol/d) and urine (8.5 ± 1.1 nmol/d ) of all rats (n = 18). In addition, butyrate production increased in all rats. In conclusion, intestinal microbiota determined 8-PN production and exposure after IX intake. Moreover, E. limosum administration increased 8-PN production in low producers, resulting in similar 8-PN production in all rats.