Analiza sekundarnih metabolita i ispitivanje farmakološke aktivnosti odabranih vrsta roda Laserpitium L. (Apiaceae)

Abstract

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 etarskom ulju herbe L. ochridanum najzastupljenija komponenta je sabinen (25,9%). U etarskom ulju cvasti L. zernyi najzastupljenije komponente su sabinen, limonen i β-felandren (18,5%, 12,0% i 12,0%, redom), a u etarskom ulju herbe L. zernyi β-pinen (20,0%). Ova dva ulja su, za razliku od drugih ispitivanih ulja vrsta roda Laserpitium, imala relativno visok sadržaj monoterpenskog alkohola terpinen-4-ola (10,6% i 12,0% u etarskom ulju herbe i cvasti L. zernyi, redom). Iz dela hloroformskog ekstrakta podzemnih organa L. latifolium rastvorljivog u metanolu, kombinovanjem pogodnih hromatografskih tehnika (hromatografija na koloni silikagela i semipreparativna HPLC) i instrumentalnih metoda za određivanje strukture (1D i 2D NMR i IR spektroskopija i HR-MS ESI+ spektrometrija), kao glavni sastojci izolovana su i okarakterisana dva daukanska estra: laserpicin (1) i acetildezoksodehidrolaserpicin (2), i dva fenilpropana: laserin (3) i latifolon (4). Uporednom HPLC analizom, u uslovima optimizovanim za razdvajanje terpenoida, uočena je velika kvalitativna sličnost hloroformskih ekstrakata podzemnih organa i herbe L. latifolium. Kvantifikacija ekstrakata izvršena je pod istim uslovima, korišćenjem izolovanih jedinjenja kao eksternih standarda. Konstatovano je da je sadržaj sva četiri jedinjenja viši u ekstraktu podzemnih organa u odnosu na ekstrakt herbe L. latifolium...

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 oils certain percent of sabinene was detected (26.8% and 14.4%, in fruit oil of L. latifolium and L. ochridanum, respectively). In herb essential oil of L. ochridanum the most abundand constituent was sabinene (25.9%). In the essential oil flower of L. zernyi sabinene, limonene and β-phellandrene were detected in higher percent (18.5%, 12.0% and 12.0%, respectively), and in oil of L. zernyi herb higher content of β-pinene (20.0%) was detected. Two latter oils, unlike the other oils, were characterised with relatively high content of terpinene-4-ol (12.0% and 10.6% in oils of L. zernyi flower and herb, respectively). From the methanol-soluble part of chloroform extract of the underground parts of L. latifolium, by combination of adequate chromatographic techniques (FC and semi-preparative HPLC) and instrumental methods for structure elucidation (1D and 2D NMR and IR spectroscopy and HR-MS (ESI+) spectrometry), as the main constituents were isolated and elucidated the two daucane esters: laserpitin (1) and acetyldesoxodehydrolaserpitin (2), as well as the two phenylpropanes: laserin (3) and latifolon (4). By comparative HPLC analysis in a system optimised for terpenoid separation, a huge qualitative similarity of chloroform extracts of the underground parts and herb of L. latifolium was observed. Quantitative analyses was conducted in the same system, using isolated compounds as external standards. It was demonstrated that all compounds were present in higher concentrations in the extract of the underground parts than in the herb extract of L. latifolium...