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Naravne snovi so spojine ali ekstrakti, ki izvirajo iz živih organizmov ter navadno izkazujejo biološko delovanje. So osnovna surovina za zdravila naravnega izvora. V farmakognoziji se ukvarjamo z njihovim izborom, karakterizacijo, identifikacijo in uporabo. Pogost pristop k odkrivanju potencialno zdravilnih naravnih snovi je preizkušanje učinkov rastlinskih izvlečkov, ki nas v povezavi s kromatografskimi separacijami vodi do izolacije biološko aktivnih molekul. Njihove kemijske strukture ugotavljamo s sodobnimi spektroskopskimi in spektrometrijskimi metodami in nadaljujemo s preizkušanjem njihovega farmakološkega delovanja. Ajda predstavlja bogat vir hranil z ugodnim vplivom na zdravje, zato njena poraba v skladu s popularnostjo zdravega načina življenja narašča. Poleg zdravju koristnih snovi pa ajda vsebuje tudi manj znane, fototoksične fagopirine, ki pod vplivom svetlobe oksidirajo biološko snov in povzročijo njene poškodbe. Za zagotavljanje varnosti ajde in njenih izdelkov je treba fagopirine vrednotiti tako s prehranskega kot s farmacevtskega vidika. Ne nazadnje gre tudi za spojine s potencialno terapevtsko uporabnostjo, na primer v fotodinamični terapiji. Razvili smo učinkovit postopek ekstrakcije fagopirinov z uporabo raztopine acetona in vode. Ugotovili smo, da so najprimernejši material za pridobivanje fagopirinov ajdovi cvetovi. Razvili smo preprosto in občutljivo metodo tekočinske kromatografije visoke ločljivosti s fluorescentno detekcijo. Metoda je primerna za analizo vzorcev ajde in kot izhodišče za razvoj preparativne metode za izolacijo fagopirinov. Ugotovili smo, da je poleg osnovne molekule fagopirina F v zeli ajde še vsaj sedem njegovih derivatov. Te derivate smo kromatografsko ločili na posamezne frakcije. Vrednotili smo jih z UV-Vis absorpcijo, masno spektrometrijo in NMR spektroskopijo. Ugotovili smo strukturi dveh novih fagopirinov, ki se razlikujeta po skupinah, pripetih na naftodiantronski obroč. Fagopirin F je že predhodno znana spojina z dvema piperidinskima obročema, fagopirin E je spojina s piperidinskim in pirolidinskim obročem, fagopirin A pa je spojina z dvema pirolidinskima obročema. Dokazali smo, da v ajdi ni fagopirinov, temveč zgolj protofagopirini kot njihove predstopnje z necikliziranim naftodiantronskim obročem. Po raztopitvi v ekstrakcijskem topilu in pod vplivom svetlobe se protofagopirini pretvorijo v fagopirine. Raziskovali smo topnost, fluorescenco in UV-Vis spektre fagopirinov v različnih topilih iz različnih vzorcev ajde. Za kvantitativno vrednotenje vsebnosti fagopirinov je treba pridobiti čiste spojine kot referenčne spojine. Raziskovalci namesto fagopirina kot referenčne spojine uporabljajo hipericin iz šentjanževke, ki pa zaradi nekoliko drugačne strukture in s tem drugačne absorpcije pri spektrofotometrijski detekciji ni ustrezen za točno kvantitativno vrednotenje fagopirinov. Naše ugotovitve in novo razvite metode bodo pripomogle k nadaljnjemu raziskovanju fagopirinov in vrednotenju vsebnosti in varnosti ajdovih izdelkov. V drugem delu doktorskega dela smo se ukvarjali z izvlečki debla navadne jelke. Izvlečki debel različnih vrst borovcev so znani po vsebnosti polifenolnih spojin z zanimivimi farmakološkimi učinki, predvidoma povezanimi z antioksidativnim delovanjem. Najbolj znan, raziskan in uporabljan je izvleček lubja obmorskega bora (Pinus maritima), Pycnogenol®. Ugotovili smo, da so izvlečki navadne jelke učinkovitejši antioksidanti od Pycnogenola® tako v in vitro antioksidativnih testih, kot tudi na humanih celičnih linijah, izoliranih iz periferne krvi. Komponente izvlečka lubja smo kromatografsko ločili z normalnofazno kolonsko kromatografijo pri zvišanem tlaku in reverznofazno tekočinsko kromatografijo visoke ločljivosti. Vsebovane spojine smo identificirali z metodami masne spektrometrije, UV-Vis absorpcijske spektroskopije in s primerjavo z referenčnimi spojinami. Identificirali smo šest fenolkarboksilnih kislin (galno, homovanilno, protokatehujsko, p-hidroksibenzojsko, vanilno in p-kumarno), tri flavonoide (katehin, epikatehin in katehintetrametileter) in štiri lignane (taksiresinol, 7-(2-metil-3,4-dihidroksitetrahidropiran-5-iloksi)-taksiresinol, sekolariciresinol in lariciresinol). Nadaljevali smo s farmakološkim testiranjem. Ugotovili smo, da izvleček zmanjša nastajanje ateroskleroze pri budrah na kronični aterogeni dieti. Ovrednotili smo vpliv izvlečka in dveh v njem prisotnih fenolnih kislin, p-kumarne in protokatehujske, na izolirana podganja srca, podvržena akutnim ishemijsko-reperfuzijskim poškodbam. Ugotovili smo, da je izvleček značilno zmanjšal poškodbo srčnega tkiva, merjeno preko sproščanja laktat-dehidrogenaze in zmanjšal pojavnost aritmij. p-kumarna in protokatehujska kislina nista imeli tako izrazitih učinkov, zato verjetno samo delno prispevata k sinergističnemu delovanju v izvlečku prisotnih učinkovin. Kot ustrezno surovino za raziskovanje in uporabo smo oblikovali izvleček, raztopljen v polietilenglikolu. Pojavila se je potreba po ugotavljanju vsebnosti vode v tovrstnih vzorcih. Uveljavljena in dostopna Karl-Fischer metoda z opazovanjem spremembe barve pri končni točki titracije ni bila ustrezna, saj je bil izvleček obarvan. Zato smo razvili preprosto in finančno dostopno titracijsko metodo za ugotavljanje vsebnosti vode v obarvanih vzorcih, pri kateri končno točko titracije zaznamo spektrofotometrijsko. Tako ovrednoten izdelek predstavlja dobro osnovo za nadaljnje raziskovanje in razvijanje novih prehranskih dopolnil, kozmetičnih izdelkov in zdravil. Natural products are compounds or extracts that originate from natural sources and usually exhibit biological activity. They are raw materials for natural medicines. Pharmacognosy encompasses their selection, characterization, identification and use. Bioassay-guided fractionation of plant extracts linked to chromatographic separation techniques leads to the isolation of biologically active compounds whose chemical structures can readily be determined by modern spectroscopic and spectrometric methods, followed by testing of their pharmacological activity.Buckwheat contains many healthy nutrients, and its consumption is therefore increasing. The plant contains phototoxic compounds called fagopyrins that can act as photosensitisers upon excitation with visible light causing fagopyrism, but these compounds have received little research attention. To assure adequate safety of buckwheat and its products, fagopyrins should be characterized from a nutritional and pharmaceutical point of view. Nevertheless, fagopyrins are not only health-threatening compounds but also potent photodynamic agents that deserve further research. In this work, fagopyrins extraction procedure was improved employing a mixture of acetone and water. Flowers were recognized as the best material for the extraction. A chromatographic analysis of fagopyrins was performed by developing a simple, sensitive HPLC analytical method of high-resolution employing fluorescence detection. The developed method is a good starting point for a preparative method that would enable the isolation of pure compounds. At least eight fagopyrin derivatives were observed upon isolation and were characterized via UV-Vis absorption, NMR spectroscopy and mass spectrometry. The structures of two new fagopyrins were elucidated. Fagopyrin F is a previously known compound that consists of naphthodianthrone scaffold and two piperidine moieties. Fagopyrin A differentiates by two pyrrolidine moieties instead of piperidine moieties, and fagopyrin E consists of one pyrrolidine and one piperidine moiety. The existence of protofagopyrins that originate in buckwheat and can transform into fagopyrins upon light exposure was confirmed. Information on solubility, fluorescence and UV-Vis absorbance in different solvents were obtained. The need for reference compounds of different fagopyrins that would allow proper quantitative evaluation of buckwheat samples still remains. Researchers use hypericin for that purpose, but we recognize its use as inadequate due to the different structure and consequently uncomparable absorbance in spectrophotometric detection. Our methods complement the existing knowledge regarding fagopyrins and will facilitate their further isolation and analysis of their biological activity. In the second part of our work, silver fir trunk extracts were investigated. Extracts from the bark of different conifer species are known to contain various polyphenols that possess interesting pharmacological activities. So far the most extensive research was done on the antioxidative extract of the maritime pine (Pinus maritima) bark Pycnogenol®, which is widely used in food supplements and cosmetic products. We have shown that antioxidant activity of silver fir (Abies alba) extract is higher comparing to the maritime pine bark extract in vitro and in cultured cells. Components of the extract were separated with normal phase flash chromatography and reversed phase HPLC. Structures of individual compounds were elucidated by mass spectrometry, UV-Vis absorption spectroscopy and comparison to reference compounds. Six phenolic acids were identified (gallic, homovanillic, protocatehuic, p-hydroxybenzoic, vanillic and p-coumaric), three flavonoids (catechin, epicatechin and catechin tetramethyl eter) and four lignans (taxiresinol, 7-(2-methyl-3,4-dihydroxytetrahydropyran-5-yloxy)-taxiresinol, secoisolariciresinol and laricinresinol).The research was continued with pharmacological testing. It was found that the extract exerts strong antioxidative and protective effects against atherogenic diet-induced arterial wall damage. Protective effects of the silver fir extract and its compounds as well as of two phenolic acids (p-coumaric and protocatechuic acids) were also evauated against ischemia-reperfusion-induced damage in isolated rat hearts. The silver fir extract significantly reduced tissue damage (measured as the lactate dehydrogenase release rate) and shortened the duration of arrhythmias, which was not observed in the case of phenolic acids. It was concluded that the two acids are only partially responsible for the observed effects and synergism of more compounds could be present. Silver fir extract, solubilized in polyethylene glycol was developed as an appropriate raw material for research and use. A need for water determination in such samples emerged. The most accessible technique for water content determination is Karl-Fischer titration with visual detection of a titration endpoint, which was unfortunately not suitable for the analysis of the silver fir extract due to its strong colour. Therefore an appropriate method for moisture determination in coloured samples was developed employing simple equipment and spectrophotometric detection of a titration endpoint. Silver fir extract, characterized in such manner is a suitable ingredient for development of food supplements, cosmetic products and medicines.

Leptin je adipocitni hormon proteinske narave (adipokin), ki ima pomembno vlogo pri uravnavanju energijskega ravnovesja organizma. Njegova koncentracija v plazmi je odvisna od količine belega maščevja in sprememb energijskega statusa organizma. Ob prekomernem vnašanju hrane namreč koncentracija leptina naraste, ob stradanju pa upade, kar preko izraţenih leptinskih receptorjev zaznajo specifični nevroni v hipotalamusu. Ti preko projekcij v številne ostale centre v moţganih aktivirajo različne kompenzatorne mehanizme za ponovno vzpostavitev energijskega ravnovesja. Tako leptin predstavlja najpomembnejši del povratne zanke uravnavanja količine energijskih zalog v obliki maščobnega tkiva. Pri večini debelih ljudi so plazemske koncentracije leptina povišane, kljub temu pa ne opazimo opisanih centralnih učinkov leptina za spodbujanje porabe energijskih preseţkov, kar razlagamo s pojavom neodzivnosti na leptin (leptinske rezistence) ob trajajoči debelosti. Povišane plazemske koncentracije leptina (hiperleptinemija) vodijo v prekomerno signalizacijo leptina na periferiji, kjer številne celice izraţajo funkcionalne leptinske receptorje. Vse več je dokazov, da prekomerna periferna aktivnost leptina pomembno vpliva na pojav ali poslabšanje resnih, z debelostjo povezanih obolenj, kot so nekatere oblike raka, avtoimunskih ali kardiovaskularnih bolezni. Zaradi tega narašča zanimanje za razvoj antagonistov leptina, ki bi jih kot popolnoma novo skupino učinkovin lahko uporabili pri zdravljenju omenjenih bolezni. Po drugi strani bi bili tudi agonisti leptina uporabni za nadomestno zdravljenje lipodistrofije, hipotalamične amenoreje in splošne imunske pomanjkljivosti zaradi podhranjenosti, zadnje raziskave pa nakazujejo tudi moţnost uporabe agonistov v podporni terapiji sladkorne bolezni tipa 2.Ker ima tako hiperleptinemija na eni kot pomanjkanje učinkov leptina na drugi strani škodljive posledice, smo se v okviru doktorskega raziskovalnega dela odločili s presejanjem bakteriofagnih predstavitvenih knjiţnic poiskati biološko aktivne peptidne ligande leptina ali leptinskega receptorja, ki bi predstavljali vodnice za razvoj potencialnih terapevtsko uporabnih antagonistov ali agonistov leptina. Tudi ligande leptina ali leptinskega receptorja brez biološke aktivnosti bi lahko uporabili kot detekcijske molekule v analiznih ali diagnostičnih metodah.S pomočjo presejanja bakteriofagnih knjiţnic krajših naključnih peptidov nismo uspeli izolirati ligandov leptina ali leptinskega receptorja. V afinitetnih selekcijah smo s ciljanjem nevtralizacijskih protiteles proti leptinu sicer identificirali krajše mimotope leptina, a ti niso izkazovali navzkriţne reaktivnosti z leptinskim receptorjem. Glede na razširjeno analizo rezultatov predhodnih afinitetnih selekcij, opravljenih v našem laboratoriju, sklepamo, da ima tarčna molekula, ki jo uporabimo v selekcijskem postopku, pomemben vpliv na izid slednjega v smislu uspešne izolacije krajših peptidnih ligandov. Po naših ugotovitvah so najbolj primerne tarče za selekcijo krajših peptidnih ligandov tiste, katerih naravni vezavni partnerji so peptidi oz. proteini in ki imajo na površini reţe ali ţepe, primerne za sidranje krajših peptidov. Proteini, udeleţeni v interakcije protein-protein, za katere so praviloma značilne večje in ploščate stične površine, so se namreč izkazali kot manj primerne tarče za selekcijo peptidnih ligandov. V nadaljevanju smo zato v afinitetnih selekcijah proti leptinskemu receptorju uporabili sintezne bakteriofagne predstavitvene knjiţnice enoveriţnih variabilnih fragmentov protiteles (scFv). Izolirali smo štiri različne fragmente scFv, ki specifično prepoznajo nativno obliko zunajcelične regije človeškega leptinskega receptorja, pri vezavi nanjo pa tekmujejo z leptinom. Eden od fragmentov scFv (3L5) izkazuje zmerno selektivnost do človeškega leptinskega receptorja v primerjavi z mišjim, poleg tega je v preliminarnih testih z uporabo monocitne celične linije THP-1 zaviral z leptinom spodbujeno proliferacijo celic in tako deloval kot antagonist leptina. Ocenjeni vrednosti konstant IC50 za inhibicijo vezave leptina na receptor s 3L5 in Kd za interakcijo 3L5 z receptorjem v raztopini se nahajata v nanomolarnem območju. Na podlagi tega menimo, da fragment scFv 3L5 predstavlja vodnico za nadaljnji razvoj terapevtsko uporabnih antagonistov leptina ali molekularnih sond za zaznavanje leptinskega receptorja. V zadnjem delu smo se lotili razvoja modelnega sistema za predstavitev dveh različnih proteinov na površini nitastih bakteriofagnih delcev. Pripravili smo tri različne pomoţne bakteriofage tipa 88 in primerjalno ovrednotili njihovo primernost za uvedbo zelenega fluorescenčnega proteina na kapsido fagmidnih virusnih delcev, ki obenem na površini izraţajo tudi fragmente scFv. Slednji so namenjeni prepoznavi določene tarče, zeleni fluorescenčni protein pa bi omogočal detekcijo na tarčo vezanih virionov. Uspešno predstavitev obeh funkcionalnih proteinov na rekombinantnih virusnih delcih smo potrdili s pomočjo fluorescenčne mikroskopije, pri čemer smo pod mikroskopom opazovali sefarozne delce s kovalentno vezano tarčno molekulo po inkubaciji z bifunkcionalnimi virioni. Pripravljeni pomoţni bakteriofagi ne omogočajo uvedbe zadostnega števila kopij fluorescenčnega proteina na virusne delce, da bi bili ti uporabni kot detekcijske komponente pri fluorescenčnoimunskem testu. Vseeno opisan modelni sistem predstavlja osnovo za razvoj bolj občutljivih detekcijskih komponent na osnovi bakterifagnih delcev, ki bi jih lahko vključili v nove analizne in diagnostične metode, na primer za določanje nivoja izraţanja leptinskega receptorja na celicah z imunofluorescenčnimi ali imunokemijskimi tehnikami. V okviru doktorske disertacije smo torej odkrili kompetitivne fragmente scFv proti leptinskemu receptorju in ovrednotili njihovo aktivnost v pogojih in vitro ter s tem v nabor kandidatov za razvoj terapevtsko uporabnih antagonistov leptina prispevali novo vodnico. Obenem lahko opisani fragmenti protiteles postanejo del molekularnih sond za detekcijo leptinskih receptorjev na površini celic, kar bi predstavljalo osnovo za razvoj novih diagnostičnih testov na principu določanja stopnje izraţanja leptinskega receptorja. Slednji je prekomerno izraţen na celicah določenih vrst tumorjev, v aterosklerotičnih plakih in prizadetih tkivih pri avtoimunskih boleznih. Leptin is an adipocyte-derived protein hormone (adipokine) with important role in energy balance regulation. Its plasma concentration depends on the amount of white adipose tissue and is also influenced by changes in the energy status of the organism. During the periods of excessive food intake plasma leptin levels increase while they decrease with fasting. Such changes are sensed by specific leptin-receptor expressing hypothalamic neurons, which project to many other centers in the brain to activate several compensatory mechanisms that rebalance organism’s energy status. Therefore, leptin represents the most important factor of the feedback loop which regulates the amount of energy reserves in the form of fat. Despite its elevated plasma concentrations in most obese people leptin fails to activate central compensatory mechanisms to reduce food intake and increase energy expenditure due to central leptin resistance. Increased plasma leptin concentration (hyperleptinemia) leads to excessive peripheral leptin signaling as leptin receptors are also present on diverse cells outside the central nervous system. There is accumulating evidence that excessive peripheral leptin activity contributes to the onset and progression of serious obesity-related conditions, such as certain types of cancer, autoimmune and cardiovascular diseases. In light of these findings, there is a growing interest in the development of leptin antagonists as potential novel class of drugs for use in therapy of aforementioned diseases. On the other hand, leptin agonists also hold potential as therapeutics for lipodystrophy, hypothalamic amenorrhea and general immune deficiency due to starvation. Moreover, recent studies also support the use of leptin agonists in therapy of type 2 diabetes. Because both, hyperleptinemia and leptin deficiency can have harmful consequences, we decided to screen phage display libraries with the main goal to identify biologically active peptide ligands of leptin or leptin receptor. Such peptides would be considered leads for development of therapeutically useful leptin agonists or antagonists. Even biologically inactive leptin or leptin receptor ligands might still find use as detection molecules in analytical or diagnostic techniques. We were unable to isolate any leptin or leptin receptor ligands from phage display libraries of random short peptides. While affinity selections towards anti-leptin neutralizing antibodies did yield binders, none of the mimotopes showed cross-reactivity with leptin receptor. Based on extended analysis of results from previous affinity selections performed in our laboratory, we conclude that the nature of target molecule critically affects the outcome of biopanning in terms of successful isolation of specific short peptide ligands. According to our findings, the most suitable targets for selection of short peptide ligands are those whose natural binding partners are peptides or proteins, and that contain specific topological features (e.g., clefts or pockets) allowing anchoring of short peptides. In contrast, proteins engaged in protein-protein interactions, which are generally characterized by relatively large and flat contact surfaces, proved to be less suitable targets for selection of short peptide ligands from phage display libraries. Therefore, we focused on screening phage display libraries of synthetic single-chain variable fragments (scFvs) instead. Through biopanning we isolated four different scFvs which specifically recognize the native form of extracellular region of the human leptin receptor, and compete with leptin for binding to the receptor. One of them (3L5) showed moderate selectivity for human leptin receptor as compared to its murine orthologue. In addition, 3L5 scFv inhibited leptin-stimulated proliferation of THP-1 cells in preliminary assay indicating it acts as leptin antagonist. Estimated values of the constants IC50 (for 3L5 scFv-mediated inhibition of leptin binding to the leptin receptor) and Kd (for 3L5 scFv-leptin receptor interaction in solution) are in the nanomolar range. Therefore, we believe that 3L5 scFv represents a promising lead for further development of therapeutically useful antibody-based leptin antagonists and molecular probes for leptin receptor detection. Last but not least, we developed a model system for simultaneous presentation of two different proteins on the surface of filamentous phage virions. We designed and constructed three different type 88 helper phages and evaluated their ability to introduce green fluorescent protein into the capsid of phagemid-based viral particles simultaneously expressing scFv fragments on their surface. scFvs were chosen to allow the recognition of specific target molecules, while green fluorescent protein would allow detection of target bound virions. Successful presentation of both functional proteins on recombinant viral particles was confirmed by fluorescence microscopy, where sepharose particles with covalently bound target protein were observed under the microscope after incubation with recombinant bifunctional virions. However, helper phages at this stage do not allow for the introduction of fluorescent protein onto the phage particles in sufficient copy number that would enable their use as a detection component in fluorescence-linked immunosorbent assay (FLISA). Nevertheless, the described prototype represent the basic platform for further development of more sensitive phage particle-based detection probes for analytical and diagnostic purposes, such as determining the abundance of leptin receptor on cells by immunofluorescence and immunochemical techniques. Taken together, we have identified four competitive scFv fragments targeting leptin receptor, thereby contributing new leads to the collection of candidates for the development of therapeutically useful leptin antagonists. In addition, the described scFvs can be used as components of molecular probes for detection of leptin receptors on cell surface, thus forming the basis for development of novel diagnostic tests based on determining expression level of leptin receptor. The latter is overexpressed on cells of certain types of tumors, atherosclerotic plaques and the affected tissues in autoimmune disorders.