Metabolomička studija balkanskih vrsta roda Amphoricarpos Vis. (Asteraceae)

Abstract

Metabolomika je nova naučna multidisciplinarna oblast koja obuhvata različiteaspekte biologije, hemije i matematike. Koristi moderne spektroskopske ihromatografske tehnike (NMR, IC, UV, MS, GC, LC) i multivarijantnu statističkuanalizu podataka sa ciljem da se izmere kvantitativno i kvalitativno svi metaboliti uispitivanom organizmu i na taj način dobije jasna metabolomička slika pod ispitivanimuslovima. Primena metabolomike nije ograničena samo na biljni svet, već se možeprimeniti na sve žive organizme, pa tako i na ljude. Posebno je značajna mogućnostprimene u medicini kao dijagnostičke metode ili u farmakognoziji za otkrivanje novihlekovitih sirovina. U ovoj disertaciji primena metabolomike na endemski biljni rodAmphoricarpos Vis. imala je dva cilja: (a) sticanje novih saznanja o mogućnostiprimene dobijenih rezultata u diferencijaciji balkanskih vrsta ovog roda i (b) razvojmetode 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 taksonaovog roda, koji nije do kraja raščišćen, primenjena je metabolomička studija. Po Blečićui Mayeru1 postoje tri taksona: dve vrste A. neumayeri Vis. i A. autariatus Blečić etMayer, koja se deli na dve podvrste - ssp. autariatus i ssp. bertisceus Blečić et Mayer. Sdruge strane, Webb2 priznaje samo jednu vrstu - A. neumayeri Vis., koja sadrži dvepodvrste - ssp. neumayeri i ssp. murbeckii Bošnjak. U novije vreme, na osnovuispitivanja 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), auzorci su sakupljeni na različitim lokalitetima u Crnoj Gori u fazi cvetanja. Primenjenaje 1H NMR spektroskopija kombinovana sa multivarijantnom analizom podataka.Pokazano je da je OPLS-DA (engl. Orthogonal Partial Least Squares-DiscriminantAnalysis) najefikasniji metod za diskriminaciju ispitivanih uzoraka na osnovumetabolita prisutnih u ekstraktima. Utvrđeno je da je glavna razlika između A.autariatus i A. neumayeri prisustvo karakterističnih seskviterpenskih laktonagvajanolidnog tipa (amforikarpolida). Glavni seskviterpenski lakton vrste A. neumayerije oksidovan u položaju 2, dok laktoni prisutni u obe podvrste A. autariatus suoksidovani u poziciji 9, što potvrđuje da seskviterpenski laktoni mogu bitihemotaksonomski markeri u porodici Asteraceae...

Metabolomics is a new scientific multidisciplinary field that includes variousaspects of biology, chemistry and mathematics. It uses modern spectroscopic andchromatographic techniques (NMR, IC, UV, MS, GC, LC) and multivariate statisticaldata analysis to measure quantitatively and qualitatively all of the metabolites in the testorganism and thereby obtain a clear metabolic profile under test conditions. The use ofmetabolomics is not limited to the plant world, but can be applied to all livingorganisms as well as humans. A particularly important possibility is the application inmedicine as a diagnostic method or in pharmacognosy for the discovery of new naturalsources of drugs. In this thesis, the application of metabolomics to the endemic plantgenus Amphoricarpos Vis. was applied with two objectives: (a) acquiring newknowledge of the systematization of the genus, and (b) developing a method for rapiddetection of new pharmacologically active ingredients in plant material.Genus Amphoricarpos Vis. is an endemic of the western part of the BalkanPeninsula. In order to gain more insight into the taxonomic status of the genus that isstill ambiguous, a metabolomic study was applied. Blečić and Mayer1 proposed threetaxa: two species A. neumayeri Vis. and A. autariatus Blečić et Mayer, the latter dividedinto 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 genomeexamination of the taxa from the West Balkans, only one species was proposed, A.neumayerianus (Vis.) Greuter.3In this dissertation all three taxa (according to Blečić and Mayer)1 were analysed. Thesamples 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 tobe the most effective method for discrimination of the tested samples, based on themetabolites present in the extracts. It was found that the main difference between A.autariatus and A. neumayeri was the presence of characteristic guaianolidesesquiterpene lactones (amforicarpolides). The major sesquiterpene lactones in theextract of A. neumayeri were oxidized in position 2, while the lactones present in bothsubspecies of A. autariatus were oxidized at position 9, which confirms thatsesquiterpene lactones may be chemotaxonomic markers in the Asteraceae family...