Isolation of natural hop compound and pKa determination

Navadni hmelj oziroma Humulus lupulus je dvodomna zelnata trajnica. Industrijsko ga pridelujemo zaradi storžkov, ki v lupulinskih žlezah vsebujejo mnoge za pivovarstvo in medicinsko pomembne spojine. Zaradi njih je hmelj zanimiv za razvoj prehranskih dodatkov in zdravil za inhibicijo sodobnih bolezni. Takšne so npr. α-kisline, ki jih bomo poleg ksantohumola v nalogi izolirali. Da je spojina sploh potencialno uporabna kot zdravilo oziroma prehranski dodatek, je potrebno vedeti kako jo bo organizem sprejel in metaboliziral. To proučuje farmakokinetika, na katero vpliva tudi pomembna fizikalno kemijska lastnost imenovana konstanta disociacije (pKa). Vrednost te konstante nam pove, kakšna je protoniranost molekul pri različnih pH vrednostih. Poznavanje te konstante je prikladno ne samo za področje farmakokinetike, ampak tudi za optimizacijo pogojev shranjevanja molekul ter za izračune v računski kemiji. V nalogi smo izolirali α-kislino imenovano kohumulon ter ji določili pKa vrednosti. Na začetku smo iz hmeljevih storžkov ekstrahirali ekstrakt. Iz ekstrakta smo nato s tehnikami hitre in preparativne kromatografije ločili kohumulon. Po vsaki ločbi smo čistost spremljali z analiznim HPLC. Dobili smo 460 mg kohumulona 92 % kromatografske čistosti pri valovni dolžini 280 nm. Izoliranemu kohumulona smo z UV-Vis spektrofotometrom pomerili spekter pri različnih pH vrednostih. Pri nižjih pH vrednostih je zaradi slabe topnosti kohumulona v vodi prišlo do obarjanja. Poskus smo ponovili z 10 % dodatkom metanola in tako rahlo izboljšali topnost. Meritvam smo na podlagi teoretično izpeljane enačbe prilegali modelne vrednosti in kot rezultat dobili pKa vrednosti. pKa1 vrednost smo v vodni raztopini določili kot 3,9ob dodatku metanola pa 4,2. Drugo pKa2 pa smo določili v vodni raztopini kot 10,9. Po naših podatkih smo sploh prvi, ki smo določili pKa2 vrednost kohumulonu.Common hop or Humulus lupulus is a dioecious herbaceous perennial. It is grown industrially for the cones, which in the lupulin glands contain many compounds important for brewing and medicine. Because of them, hops are interesting for the development of nutritional supplements and drugs for the inhibition of modern diseases. Such are, for example, α-acids, which we will isolate in addition to xanthohumol. In order for a compound to be potentially useful as drug or food supplement, it is necessary to know how the organism will accept and metabolize it. This is studied by pharmacokinetics, which is also influenced by an important physicochemical property called the dissociation constant (pKa). The value of the constant tells us the protonation of the molecule at different pH values. Knowing the constant is convenient not only for the field of pharmacokinetics, but also for the optimization of storage conditions of molecules and for calculations in computational chemistry. In the assignment, we isolated an α-acid called cohumulone and determined its pKa values. At the beginning, we extracted an extract from the hop cones. Cohumulone was then separated from the extract using flash and preparative chromatography techniques. We obtained 460 mg of cohumulone with 92% chromatographic purity at a wavelength of 280 nm. Purity was monitored by analytical HPLC after each separation. The spectrum of the isolated cohumulone was measured with a UV-Vis spectrophotometer at different pH values. At lower pH, precipitation occurred due to the poor solubility of cohumulone in water. The experiment was repeated with the addition of 10% methanol, thus slightly improving the solubility. Based on the theoretically derived equation, model values were fitted to the measurements and pKa values were obtained as a result. The pKa1 value in aqueous solution was determined as 3.9and 4.2 with the addition of methanol respectively. Another pKa2 was determined in aqueous solution as 10.9. According to our best knowledge, we are the first to determine the pKa2 value of cohumulone