| Abstract | Biljni svijet je prirodno skladište bioaktivnih tvari. Jedna od najviše istraženih skupina svakako su flavonoidi koji imaju višestruku funkciju u biljkama, ali i pozitivan utjecaj na ljudsko zdravlje. Flavonoidi u biljkama mogu postojati u slobodnom obliku, ali najčešće su glikolizirani, metilirani, acetilirani, prenilirani ili polimerizirani, što utječe i na njihovu bioaktivnost. Flavonoidni dimeri, poznatiji pod nazivom biflavonoidi, se sastoje od dva identična ili različita flavonoidna monomera. U odnosu na monomere značajno su manje istraženi iako pokazuju bolju biološku aktivnost od njih i nedavno je prepoznat njihov farmakološki potencijal, ponajviše zbog antivirusne, antikancerogene i antifungalne aktivnosti. Biflavonoidi su prisutni u biljkama koje se koriste u tradicionalnoj medicini poput ginka (Ginkgo biloba L.). Kako bi se biflavonoidi mogli koristiti, potrebno ih je izolirati iz prirodnih izvora. Ovaj proces obično uključuje korištenje raznih organskih otapala, koja mogu biti opasna za okoliš i ljude stoga se u zadnje vrijeme sve više koriste eutektična otapala (engl. Deep Eutectic Solvent, DES) koja su prepoznata kao zelena i jeftina ekstrakcijska sredstva, a dodatna prednost im je izražena fleksibilnost. Osim toga, zbog svoje netoksičnosti i biorazgradivosti odgovaraju svim zahtjevima i principima zelene kemije. Zbog svega navedenog, DES se smatraju vrlo pogodnima za izolaciju biomolekula kao što su biflavonoidi. U sklopu ovog rada, prirodna eutektička otapala (engl. Natural Deep Eutectic Solvent, NADES) korištena su za ekstrakciju biološki aktivnih molekula iz listova ginka. Pomoću računalnog modela COSMO-RS ispitana je topljivost ginkgetina, najčešćeg predstavnika biflavonoida u listovima ginka, i amentoflavona, najpolarnijeg biflavonoida u ginku. Na temelju topljivosti procijenjeno je kako bi 15 od 250 ispitanih NADES moglo biti dobro ekstrakcijsko sredstvo. Odabrani NADES su u sljedećem koraku sintetizirani i karakterizirani (gustoća, viskoznosti, pH-vrijednost). U slijedu šaržnih ekstrakcija pomoću odabranih 15 otapala pri istim procesnim uvjetima (temperatura, vrijeme ekstrakcije i omjer početne mase usitnjenog lišća ginka i volumena otapala) ispitana je učinkovitost ekstrakcije biflavonoida, ukupnih flavonoida, polifenola i fenolnih kiselina. Najveća učinkovitost ekstrakcije ukupnih flavonoida postignuta je pomoću NADES betain:etilen glikol (B:EG) 1:2 s 30 % H2O (w/w). Dobiveni rezultati uspoređeni su s ekstrakcijom provedenom pod istim uvjetima korištenjem 80 % metanola kao otapala, pri čemu je korištenjem odabranog NADES postignuta veća učinkovitost ekstrakcije za 24 %.
U sljedećem koraku, ekstrakcija s odabranim NADES je dodatno optimirana korištenjem eksperimentalnog Box-Behnken dizajna na tri razine s tri faktora: vrijeme ekstrakcije, omjer početne mase usitnjenog lišća i volumena NADES-a i temperatura. Dobiveni rezultati pokazali su da su optimalni uvjeti ekstrakcije korištenjem odabranog NADES: vrijeme ekstrakcije
45 min, omjer početne mase usitnjenog lišća i volumena NADES 20 mg/mL, te temperatura 20 ºC. Naposljetku, korištenjem HPLC-DAD metode u ekstraktima su određene koncentracije 5 najzastupljenijih biflavonoida u lišću ginku (amentoflavon, ginkgetin, izoginkgetin, bilobetin i sciadopitisin). |
| Abstract (english) | The plant kingdom is a known reservoir of bioactive natural products. The best-studied group to date is flavonoids, which perform multiple functions in plants and have positive impact on human health. Flavonoids can occur in free form in plants, but are more commonly glycosylated, methylated, acetylated, prenylated, or polymerized, which affects their bioactivity. Flavonoid dimers, known as biflavonoids, are consist of two identical or different flavonoid units. They are a less-studied subgroup of flavonoids, although they have bigger bioactivity, and they have recently been recognized as potential pharmaceutical agents, particularly because of their antiviral, anticancer, antifungal, and other bioactivities. They are commonly found in plants used in traditional medicine, such as ginkgo (Ginkgo biloba L.). The crucial step for the pharmaceutical use of any natural product is its isolation from the natural matrix. This process usually involves the use of various organic solvents, which can be hazardous to the environment and humans. Therefore, deep eutectic solvents (DES) have recently been recognized as environmentally friendly and cheap extraction agents, whose additional advantage is their pronounced flexibility. Besides that, there are non-toxic and biodegradable, so they satisfy all the requirements and principles of green chemistry. Due to all of the above, DES are considered very suitable for the isolation of biomolecules such as biflavonoids. As part of this work, natural deep eutectic solvents (NADES) were used to extract biologically active molecules from ginkgo leaves. To select the best NADES for efficient extraction of biflavonoids from ginkgo, COSMOtherm software was used, and solubility of ginkgetin, as the most common biflavonoid in ginkgo leaves, and amentoflavone, as the most polar, was calculated. Based on the obtained solubility predictions, 15 of 250 tested NADES could be effective in extraction of biflavonoids. In the next step, those 15 NADES were synthesized, and characterized (density, viscosity, and pH). After synthesis, the extraction of biflavonoids, total phenols, flavonoids and phenolic acids was performed in a series of batch experiments with the same process conditions (temperature, extraction time and ratio of initial mass of shredded leaves and mass of NADES) where the extraction efficiency of total flavonoids was defined as a criterion for the selection of the best NADES. The highest extraction efficiency was obtained with Betaine:Ethylene Glycol (B:EG) 1:2 NADES with 30 % H2O (w/w). Compared to extraction with 80 % methanol, 24 % higher extraction efficiency was obtained using B:EG 1:2 with 30 % H2O (w/w) NADES under the same process conditions. In addition, extraction with that solvent was optimized using the experimental Box-Behnken design at three levels with three factors: extraction time, ratio of initial mass of shredded leaves and mass of NADES, and temperature. It was concluded that the optimal extraction conditions are: extraction time of 45 min, the ratio of the initial mass of shredded leaves and mass of NADES 20 mg/mL, and temperature 20 ºC. Finally, the concentrations of the 5 most abundant biflavonoids in ginkgo (amentoflavone, ginkgetin, isoginkgetin, bilobetin and sciadopitisin) were determined using HPLC-DAD method. |
