The metabolomic study of the Balkan species of the genus Amphoricarpos Vis. (Asteraceae)

Metabolomika je nova naučna multidisciplinarna oblast koja obuhvata različite aspekte biologije, hemije i matematike. Koristi moderne spektroskopske i hromatografske tehnike (NMR, IC, UV, MS, GC, LC) i multivarijantnu statističku analizu podataka sa ciljem da se izmere kvantitativno i kvalitativno svi metaboliti u ispitivanom organizmu i na taj način dobije jasna metabolomička slika pod ispitivanim uslovima. Primena metabolomike nije ograničena samo na biljni svet, već se može primeniti na sve žive organizme, pa tako i na ljude. Posebno je značajna mogućnost primene u medicini kao dijagnostičke metode ili u farmakognoziji za otkrivanje novih lekovitih sirovina. U ovoj disertaciji primena metabolomike na endemski biljni rod Amphoricarpos Vis. imala je dva cilja: (a) sticanje novih saznanja o mogućnosti primene dobijenih rezultata u diferencijaciji balkanskih vrsta ovog roda i (b) razvoj metode za brzo otkrivanje novih farmakološki aktivnih sastojaka u biljnom materijalu. Vrste roda Amphoricarpos Vis. rasprostranjene su na Balkanskom poluostrvu. Anatoliji i Kavkazu. Da bi se dobilo više uvida u taksonomski status balkanskih taksona ovog roda, koji nije do kraja raščišćen, primenjena je metabolomička studija. Po Blečiću i Mayeru1 postoje tri taksona: dve vrste A. neumayeri Vis. i A. autariatus Blečić et Mayer, koja se deli na dve podvrste - ssp. autariatus i ssp. bertisceus Blečić et Mayer. S druge strane, Webb2 priznaje samo jednu vrstu - A. neumayeri Vis., koja sadrži dve podvrste - ssp. neumayeri i ssp. murbeckii Bošnjak. U novije vreme, na osnovu ispitivanja genoma vrsta ovog roda predložena je samo jedna vrsta, А. neumayerianus (Vis.) Greuter.3 U ovom radu analizirana su sva tri taksona (prema Blečiću i Mayeru), a uzorci su sakupljeni na različitim lokalitetima u Crnoj Gori u fazi cvetanja. Primenjena je 1H NMR spektroskopija kombinovana sa multivarijantnom analizom podataka. Pokazano je da je OPLS-DA (engl. Orthogonal Partial Least Squares-Discriminant Analysis) najefikasniji metod za diskriminaciju ispitivanih uzoraka na osnovu metabolita prisutnih u ekstraktima. Utvrđeno je da je glavna razlika između A. autariatus i A. neumayeri prisustvo karakterističnih seskviterpenskih laktona gvajanolidnog tipa (amforikarpolida). Glavni seskviterpenski lakton vrste A. neumayeri je oksidovan u položaju 2, dok laktoni prisutni u obe podvrste A. autariatus su oksidovani u poziciji 9, što potvrđuje da seskviterpenski laktoni mogu biti hemotaksonomski markeri u porodici Asteraceae...Metabolomics is a new scientific multidisciplinary field that includes various aspects of biology, chemistry and mathematics. It uses modern spectroscopic and chromatographic techniques (NMR, IC, UV, MS, GC, LC) and multivariate statistical data analysis to measure quantitatively and qualitatively all of the metabolites in the test organism and thereby obtain a clear metabolic profile under test conditions. The use of metabolomics is not limited to the plant world, but can be applied to all living organisms as well as humans. A particularly important possibility is the application in medicine as a diagnostic method or in pharmacognosy for the discovery of new natural sources of drugs. In this thesis, the application of metabolomics to the endemic plant genus Amphoricarpos Vis. was applied with two objectives: (a) acquiring new knowledge of the systematization of the genus, and (b) developing a method for rapid detection of new pharmacologically active ingredients in plant material. Genus Amphoricarpos Vis. is an endemic of the western part of the Balkan Peninsula. In order to gain more insight into the taxonomic status of the genus that is still ambiguous, a metabolomic study was applied. Blečić and Mayer1 proposed three taxa: two species A. neumayeri Vis. and A. autariatus Blečić et Mayer, the latter divided into two subspecies, ssp. autariatus i ssp. bertisceus Blečič et Mayer. On the other hand, Webb2 recognizeed only one species, A. neumayeri Vis. divided into two subspecies, ssp. neumayeri i ssp. murbeckii Bošnjak. Recently, on the basis of a genome examination of the taxa from the West Balkans, only one species was proposed, A. neumayerianus (Vis.) Greuter.3 In this dissertation all three taxa (according to Blečić and Mayer)1 were analysed. The samples were collected at different locations in Montenegro at the stage of flowering. 1H NMR spectroscopy combined with multivariate data analysis was applied. Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) has been shown to be the most effective method for discrimination of the tested samples, based on the metabolites present in the extracts. It was found that the main difference between A. autariatus and A. neumayeri was the presence of characteristic guaianolide sesquiterpene lactones (amforicarpolides). The major sesquiterpene lactones in the extract of A. neumayeri were oxidized in position 2, while the lactones present in both subspecies of A. autariatus were oxidized at position 9, which confirms that sesquiterpene lactones may be chemotaxonomic markers in the Asteraceae family..