| Abstract | U okviru doktorskog rada provedena su ispitivanja antimikrobnog, protuupalnog te antioksidacijskog djelovanja ekstrakata
vrste Erodium cicutarium (L.) L’Hér. ex Aiton, Geraniaceae u uvjetima in vitro. Ispitivanja navedenih aktivnosti praćena su
opsežnom fitokemijskom analizom ektrakata i eteričnog ulja. Antimikrobna aktivnost iscrpina poglavito je izražena na
sojeve vrste Staphylococcus aureus, uključujući i kliničke MRSA sojeve. Uz pokazanu kinetiku bakteriostatskog i
baktericidnog učinka u vremenu te anti-biofilm aktivnost, bioautografijom je uz semipreparativnu izolaciju i UHPLC-LTQ
OrbiTrap MS4 analizu, kao vrhunac ovog doktorskog rada, provedena izolacija i karakterizacija antimikrobno aktivnih
frakcija. Dobiveni rezultati ukazuju na derivate galne kiseline kao spojeve poglavito odgovorne za opaženi antimikrobni
učinak. Među njima je najzastupljeniji spoj galoil šikiminska kiselina. Poseban je naglasak i na 7 spojeva iz antimikrobno
aktivnih frakcija, koji su po prvi puta unutar ovog doktorskog rada identificirani za vrstu E. cicutarium, rod Erodium, te
porodicu Geraniaceae. To su narcisin, galoil pentozid izomer 1 i izomer 2, metilgaloil-kafeoil heksozid, metilgaloil-kumaroil
heksozid, te izomer 1 i izomer 2 trimetilelagične kiseline. Protuupalni potencijal vrste E. cicutarium u uvjetima in vitro
ponajprije je pokazan kroz rezultate izravne inhibicije enzima COX1 i COX2 i inhibiciju citokina IL-6, te inhibiciju lučenja
dušik (II) oksida na modelu THP-1 stanica. Antioksidacijsku aktivnost u uvjetima in vitro, ispitanu metodama DPPH, ABTS,
FRAP, CUPRAC te metodom izbjeljivanja β-karotena, pokazali su svi ispitani uzorci u svim provedenim pokusnim
uvjetima. Detaljan polifenolni profil vrste E. cicutarium utvrđen je metodom UHPLC-LTQ OrbiTrap MS4. Analizirane su
metanolne i vodene iscrpine ove vrste s četiri lokaliteta na području Republike Hrvatske (Podvinje, Plitvice, Trešnjevka,
Buzin), a identificirano je ukupno 85 sastavnica. Najzastupljeniji su derivati galne kiseline (24 sastavnice), derivati elagične
kiseline – uključujući elagitanine (22 sastavnice), 19 flavonolnih glikozida, 8 derivata fenolnih kiselina, 7 derivata drugih
hidroksibenzojevih kiselina, 3 flavonolna aglikona te 2 procijanidina. Nagasak je na 25 identificiranih sastavnica, koje do
sada nisu pronađene unutar roda Erodium, te porodice Geraniaceae. Od ukupno 17 kvantificiranih sastavnica metodom
UHPLC-QqQ-MS/MS provedena je kvantifikacija sastavnica, među kojima je najzastupljenija galna kiselina. GC-MS
analizom eteričnog ulja ukupno je identificirano 50 spojeva, s ugljikovodicima kao glavnom skupinom spojeva
(najzastupljeniji spoj je heksadekanoična kiselina). |
| Abstract (english) | Research in this doctoral thesis was focused on in vitro antimicrobial, ani-infammatory, and antioxidative activity of
Erodium cicutarium (L.) L’Hér. ex Aiton, Geraniaceae extracts, strongly underlined with phytochemical characterization of
extracts and the essential oil. Antimicrobial activity was evident, especially against Staphylococcus aureus strains, including
MRSA. Next to observed anti-biofilm activity, the time-kill assay was performed and bactericidal and bacteriostatic activity
were studied in time. As a highlight of this work, active antimicrobial fractions were identified, isolated and extensively
phytochemically characterized using UHPLC-LTQ OrbiTrap MS4. Obtained results indicate that gallic acid derivatives
could be the compounds most responsible for the observed in vitro antimicrobial activity. Galloyl-shikimic acid was
identified as the most abundant compound among them. To stress out, 7 of the identified compounds are compounds
detected for the first time not only for E. cicutarium, but in the Geraniaceae family as well. Among them narcissin, galloyl
pentoside isomer 1 and isomer 2, methylgalloyl-caffeoyl hexoside, methylgalloyl-coumaroyl hexoside, trimethylellagic acid
isomer 1 and isomer 2. In vitro anti-inflammatory activity testing of E. cicutarium extracts showed inhibitory activity on
COX-1 and COX-2, cytokine IL-6 production, as well as on production of NO, indicating their anti-inflammatory activity.
TNF-α production was induced, showing the potential of E. cicutarium extracts for immunomodulation. In vitro antioxidant
activity was shown for all samples in all performed assays – DPPH, ABTS, FRAP, CUPRAC and the β-carotene bleaching
assay. Detailed phenolic profiling of water and methanol extracts was undertaken with UHPLC-LTQ OrbiTrap MS4 for
identification, and UHPLC-QqQ-MS/MS for quantification. Samples were collected from 4 localities in Croatia (Podvinje,
Plitvice, Trešnjevka, Buzin). In total, 85 phenolic compounds were identified. Among them mainly gallic acid derivatives
(24 compounds), ellagic acid derivatives, including ellagitannins (22 compounds), 19 flavonol glycosides, 8
hydroxycinnamic acid derivatives, 7 other hydroxybenzoic acid derivatives, 3 flavonol aglycones, and 2 procyanidins. Very
important results from this study show the presence of newly identified compounds – 25 new compounds were found, that
have not been reported for the Erodium genera or the Geraniaceae family before. Among 17 quantified components, gallic
acid stands out as the phenolic constituent with highest quantified content among all samples. The GC-MS analysis of
essential oils showed a total of 50 identified compounds. Hydrocarbons are the main class of constituents, with hexadecanoic acid as the major component. |
