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A new auronolignan, named cotinignan B, was isolated from Cotinus coggygria Scop. Structure elucidation was performed on the basis of 1H, 13C NMR, COSY, NOESY, HSQC, and HMBC experiments, supported with HR-ESI-MS, IR and UV. Additional J-HMBC NMR experiment was essential to resolve the configuration of the trisubstituted double bond. This compound represents the secondly discovered natural compound belonging to the rare auronolignan type.

A new auronolignan, named cotinignan B, was isolated from Cotinus coggygria Scop. Structure elucidation was performed on the basis of 1H, 13C NMR, COSY, NOESY, HSQC, and HMBC experiments, supported with HR-ESI-MS, IR and UV. Additional J-HMBC NMR experiment was essential to resolve the configuration of the trisubstituted double bond. This compound represents the secondly discovered natural compound belonging to the rare auronolignan type.

During 2021 and 2022, the pandemic of corona viruses (COVID-19) caused the enormous increase in use of antioxidants and supplements. The latest recommendations from the World Health Organization were to use natural sources of antioxidants which can help in boosting immunity (WHO, 2019). Dietary antioxidants from fruits and vegetables play an important role in mitigating the damaging effects of reactive oxygen species (ROS) on cellular macromolecules (lipids, proteins, and DNA) (Rao et al., 2010). The raspberries have high antioxidant phytochemical contents which can be preserved by freezedried process. The yield of bioactive compounds from plant sources depends on solvent properties, nature of the plant material and extraction temperature and time (Xue et al., 2020). In the green chemistry approaches, natural deep eutectic solvents (NADES) were typically used as a substitute for organic solvents in order to preserve the environment (Espino et al., 2016).

This study revealed results from reinvestigation of sesquiterpene lactones composition in the crude and leaf surface extracts of Amphoricarpos plants by rapid resolution liquid chromatography (RRLC) coupled with quadrupole time-of-flight mass spectrometry (QToF MS) and the tentative identification of undescribed sesquiterpene lactones is presented.

The content of nonacosan-10-ol and n-alkanes in needle waxes of 12 Pinus taxa (P. halepensis, P. heldreichii, P. mugo, P. nigra ssp. nigra, P. nigra ssp. dalmatica, P. peuce, P. pinaster, P. pinea, P. ponderosa, P. strobus, P. sylvestris and P. wallichiana) were examined. In eight Pinus taxa (except of P. halepensis, P. pinaster, P. heldreichii and P. peuce) nonacosan-10-ol contents were examined for the first time. In all examined pines C29, C27 or C25 were the main n-alkane compounds. C range was mainly 18-–33. In six Pinus taxa (P. mugo, P. nigra ssp. dalmatica, P. sylvestris, P. pinea, P. strobus and P. wallichiana) carbon preference index and average chain length values were examined for the first time. In the plane of Axis 1 (PCA), P. strobus, P. mugo and P. wallichiana were strongly separated from P. heldreichii and P. pinea. In the plane of Axis 2, P. peuce, P. strobus and P. wallichiana as well as P. heldreichii and P. pinea diverged from other examined species. In DA, P. heldreichii, P. strobus, P. peuce and P. wallichiana diversed. CA divided sections Pinus and Pinaster from the section Strobi.

Phytochemistry deals with the study of secondary metabolites produced by plants that synthesize these compounds for many reasons, including their own protection against attack of herbivores and plant diseases. Secondary metabolites are believed to represent plant adaptation to various environmental factors and that they enabled the survival of the species. Secondary metabolites of plants can have curative or toxic effects in humans and animals. Herbal medicine has a long tradition in folk medicine and until the early 20th century, when synthetic organic chemistry began to develop, plants were the main source of medicines. The two basic goals of our phytochemical research are: isolation and identification of new (biologically active) compounds ? potential drugs, and chemotaxonomy (chemosystematics). In the following text through one selected example, the genus Amphoricarpos Vis., our phytochemical research is shown on both aspects.

In leaf cuticular wax of Pinus pinaster, content of nonacosan-10-ol is high (77.1% on average). n-Alkanes ranged from C18 to C35 with the most dominant C29 (24.8%). The carbon preference index (CPItotal) ranged from 3.1 to 5.6 (4.0 on average), while the average chain length (ACLtotal) ranged from 14.0 to 17.0 (14.8 on average). Long-chain n-alkanes (n-C25-35) strongly dominated (80.1%) over middle-chain (n-C21-24= 18.9%) and short-chain (n-C18-20= 0.9%) n-alkanes.

The content of nonacosan-10-ol and n-alkanes in needle waxes of 12 Pinus taxa (P. halepensis, P. heldreichii, P. mugo, P. nigra ssp. nigra, P. nigra ssp. dalmatica, P. peuce, P. pinaster, P. pinea, P. ponderosa, P. strobus, P. sylvestris and P. wallichiana) were examined. In eight Pinus taxa (except of P. halepensis, P. pinaster, P. heldreichii and P. peuce) nonacosan-10-ol contents were examined for the first time. In all examined pines C29, C27 or C25 were the main n-alkane compounds. C range was mainly 18-–33. In six Pinus taxa (P. mugo, P. nigra ssp. dalmatica, P. sylvestris, P. pinea, P. strobus and P. wallichiana) carbon preference index and average chain length values were examined for the first time. In the plane of Axis 1 (PCA), P. strobus, P. mugo and P. wallichiana were strongly separated from P. heldreichii and P. pinea. In the plane of Axis 2, P. peuce, P. strobus and P. wallichiana as well as P. heldreichii and P. pinea diverged from other examined species. In DA, P. heldreichii, P. strobus, P. peuce and P. wallichiana diversed. CA divided sections Pinus and Pinaster from the section Strobi.

In needle cuticular wax of Pinus halepensis, nonacosan-10-ol is high (77.08% on average). n-Alkanes ranged from C18 to C35 with the most dominant C27 and C29 (32.4% and 25.8%, respectively). The carbon preference index ranged from 3.2 to 5.4 (3.4 on average), while the average chain length ranged from 14.0 to 18.0 (17.2 on average). Long-chain n-alkanes strongly dominated (95.1%).

The variability of n-alkanes in the needle cuticular wax of Pinus heldreichii and P. peuce in two natural populations from the Scardo-Pindic mountains was investigated for the first time. Gas chromatography (GC) and gas chromatography/mass spectrometry (GC-MS) analyses of two-year-old needles were performed us-ing an Agilent 7890A GC equipped with an inert 5975C XL EI/CI mass spectrometer detector (MSD) and a flame ionization detector (FID) connected by a capillary flow technology 2-way splitter with make-up. An HP-5MS capillary column was used. n-Alkanes ranged from C19 to C33. In P. heldreichii the most abundant were C23, C25 and C27 (16.3, 15.6 and 12.8 % on average, respectively), while in P. peuce they were C29, C25, and C27 (16.5, 15.3 and 13.5 % on average, resp.). Mid-chain and long-chain n-alkanes prevailed in both species. Principle component analysis (PCA) and Cluster analyses of five and six n-alkanes, respectively, showed divergence of the Scardo-Pindic populations from the Dinaric ones.

Analysis of composition of CH2Cl2 surface extract of leaves of Amphoricarpos neumayeri Vis. revealed 16 sesquiterpene lactones with guaianolide skeleton, so called amphoricarpolides, typical for this genus. Four of them, 13-16, were new derivatives and their structures were elucidated by detailed analyses of IR, NMR and MS data. Amphoricarpolide (9), its 15-O-acetyl derivative (5), and two 9 beta-hydroxyamphoricarpolides, 3,15-di-O-acetyl- and 3-O-acetyl-15-O-isovaleroyl derivatives (3 and 6, respectively) were tested for in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using cytochalasin-B blocked micronucleus (CBMN) assay. The tested compound exerted a beneficial effect by decreasing DNA damage of human lymphocytes.

Analysis of composition of CH2Cl2 surface extract of leaves of Amphoricarpos neumayeri Vis. revealed 16 sesquiterpene lactones with guaianolide skeleton, so called amphoricarpolides, typical for this genus. Four of them, 13–16, were new derivatives and their structures were elucidated by detailed analyses of IR, NMR and MS data. Amphoricarpolide (9), its 15-O-acetyl derivative (5), and two 9β-hydroxyamphoricarpolides, 3,15-di-O-acetyl- and 3-O-acetyl-15-O-isovaleroyl derivatives (3 and 6, respectively) were tested for in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using cytochalasin-B blocked micronucleus (CBMN) assay. The tested compound exerted a beneficial effect by decreasing DNA damage of human lymphocytes.

Volatiles of Thymus serpyllum L. were obtained by hydrodistillation (HD), simultaneous hydrodistillation, and extraction (SDE) and static headspace gas chromatography-mass spectrometry analysis (head space [HS]), respectively. Monoterpenes were the most dominant in all 3 techniques (84.8%-94.2%). Essential oil profiles obtained by HD and SDE were similar, with oxygenated monoterpenes being the most abundant (up to 75.4%). In HS volatiles of T. serpyllum monoterpene hydrocarbons strongly dominated (94.2%). The main compounds were α-terpinyl acetate (HD and SDE) and myrcene (HS).

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 d, 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 to

New aurone epoxide, 2,10-oxy-10-methoxysulfuretin (14), and new auronolignan (15), named cotinignan A, were isolated by silica gel column and semipreparative HPLC chromatography from the methylene chloride/methanol extract of Cotinus coggygria Scop. heartwood. In addition, thirteen known secondary metabolites namely sulfuretin, 2,3-trans-fustin, fisetin, butin, butein, taxifolin, eriodictyol, 3,5,5,7-tetrahydroxyflavanone, 3,4,7-trihydroxyflavone, 3-O-methyl-2,3-trans-fustin, 3-O-galloyl-2,3-trans-fustin, beta-resorcylic acid and 3-O-beta-sitosterol glucoside were isolated as well. Their structures were elucidated by 1D and 2D NMR, HR-ESI-MS, IR and UV. Ten out of eleven isolated flavonoids possess 7, 3 and 4 hydroxy groups. These structural features could be considered as chemotaxonomic characteristic of flavonoids from C. coggygria. Cotinignan A (15) represents new subclass of secondary metabolites - auronolignans. [GRAPHICS]

New aurone epoxide, 2,10-oxy-10-methoxysulfuretin (14), and new auronolignan (15), named cotinignan A, were isolated by silica gel column and semipreparative HPLC chromatography from the methylene chloride/methanol extract of Cotinus coggygria Scop. heartwood. In addition, thirteen known secondary metabolites namely sulfuretin, 2,3-trans-fustin, fisetin, butin, butein, taxifolin, eriodictyol, 3',5,5',7–tetrahydroxyflavanone, 3',4',7-trihydroxyflavone, 3-O-methyl-2,3-trans-fustin, 3-O-galloyl-2,3-trans-fustin, β-resorcylic acid and 3-O-β-sitosterol glucoside were isolated as well. Their structures were elucidated by 1D and 2D NMR, HR-ESI-MS, IR and UV. Ten out of eleven isolated flavonoids possess 7, 3' and 4' hydroxy groups. These structural features could be considered as chemotaxonomic characteristic of flavonoids from C. coggygria. Cotinignan A (15) represents new subclass of secondary metabolites - auronolignans. (Figure presented.).

Table S1. HPLC program for the quantification of the main compounds from the C. coggygria CH2Cl2/MeOH extract Figure S1. UV spectra of 14 in MeOH, with addition of AlCl3, and HCl Figure S2. UV spectra of compound 14 in MeOH, with NaOAc, NaOAc+H3BO3 and with NaOMe Figure S3. 1H NMR spectrum of compound 14 Figure S4. 13C NMR spectrum of compound 14 Figure S5. Aromatic part of the COSY spectrum of 14 Figure S6. Aromatic part of the NOESY spectrum of 14 Figure S7. Aromatic part of the HSQC spectrum of 14 Figure S8. Aromatic part of the HMBC spectrum of 14 Figure S9. Key HMBC correlations of 14 Table S2. 1H, 13C NMR and HMBC data of 14 (CD3OD, δ ppm, J in Hz) Figure S10. Aromatic part of the 1H NMR spectrum of 15 Figure S11. Aliphatic part of the 1H NMR spectrum of 15 Figure S12. 13C NMR spectrum of 15 Figure S13. HSQC spectrum of 15 Figure S14. The first part of the HMBC spectrum of 15 Figure S15. The second part of the HMBC spectrum of 15 Figure S16. HMBC (4 Hz) correlation H-7''/C-4' (15) Figure S17. The first part of the NOESY spectrum of 15 Figure S18. The second part of the NOESY spectrum of 15 Table S3. 1H and 13C NMR data of 15 (CD3OD, δ ppm, J in Hz) Figure S19. 3-Aryl-propanol moiety in 15 Figure S20. Key HMBC correlations in 15 Figure S21. UV spectrum of 15 Figure S22. UV spectrum of 10

The n-alkane composition in the leaf cuticular waxes of natural populations of Bosnian pine (Pinus heldreichii), Austrian pine (P.nigra), and Macedonian pine (P.peuce) was compared for the first time. The range of n-alkanes was wider in P.nigra (C-16-C-33) than in P.heldreichii and P.peuce (C-18-C-33). Species also diverged in abundance and range of dominant n-alkanes (P. heldreichii: C-23, C-27, and C-25; P.nigra: C-25, C-27, C-29, and C-23; P.peuce: C-29, C-25, C-27, and C-23). Multivariate statistical analyses (PCA, DA, and CA) generally pointed out separation of populations of P.nigra from populations of P.heldreichii and P.peuce (which were, to a greater or lesser extent, separated too). However, position of these species on the basis of n-alkane composition was in accordance neither with infrageneric classification nor with recent molecular and terpene investigations.

The n-alkane composition in the leaf cuticular waxes of natural populations of Bosnian pine (Pinus heldreichii), Austrian pine (P.nigra), and Macedonian pine (P.peuce) was compared for the first time. The range of n-alkanes was wider in P.nigra (C-16-C-33) than in P.heldreichii and P.peuce (C-18-C-33). Species also diverged in abundance and range of dominant n-alkanes (P. heldreichii: C-23, C-27, and C-25; P.nigra: C-25, C-27, C-29, and C-23; P.peuce: C-29, C-25, C-27, and C-23). Multivariate statistical analyses (PCA, DA, and CA) generally pointed out separation of populations of P.nigra from populations of P.heldreichii and P.peuce (which were, to a greater or lesser extent, separated too). However, position of these species on the basis of n-alkane composition was in accordance neither with infrageneric classification nor with recent molecular and terpene investigations.

Simultaneous hydrodistillation and extraction of Pinus cembra needles from Slovakia was done (via Likens Nickerson apparatus) for the first time. In essential oil extracts 55 compounds were identified, comprising 99.6% of the extract. The most abundant were monoterpene hydrocarbons (71.0%). In the terpene profile -pinene, limonene/-phellandrene, germacrene D, -pinene, and -cadinene dominated (53.2%, 11.4%, 9.4%, 4.6%, and 4.3%, respectively). Seven new compounds for P. cembra, such as methyl daniellate (0.5%), 1,8 cineole (0.2%) and trans-cadina-1(6),4-diene (0.2%), etc. were found. In needle cuticular wax of P. cembra the amount of nonacosan-10-ol was 75.8%. n-Alkanes ranged from C20 to C35 with the most dominant C31, C29 and C33 (33.4%, 16.9%, and 9.6%, resp.). Differences in terpene profiles between Slovakian and Greece from one side and Romanian and Polish cembran pines on the other side could be the consequence of its disjuncted areal in Carpathian Mountains caused by glaciation and survival of species in different ecological niches. Obtained differences in n-alkane profiles among our and literature results could be the consequence of different age of trees.

We examined for the first time the leaf micromorphology, phytochemistry and biological activity of the rare and stenoendemic Amphoricarpos elegans Albov (Compositae) from Georgia. Scanning electron microscopy (SEM) revealed the presence of glandular trichomes on the leaves, which appeared as glandular dots that are considered the main sites of biosynthesis and accumulation of sesquiterpene lactones. Using high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy analyses, we identify and characterized 3-deoxyamphoricarpolide, a known sesquiterpene lactone for the genus Amphoricarpos Vis. Regarding chemotaxonomic significance, 3-deoxyamphoricarpolide represents a link between Balkan and Caucasian species of the genus. The antioxidative capacity of different leaf extracts, obtained using a Soxhlet extractor, was evaluated by two radical scavenging assays: DPPH 91,1-diphenyl-2-picrylhydrazyl) radical and the 2,2-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), and ferric ion reducing antioxidant power (FRAP). The total phenolic and flavonoid contents were also determined. The highest antioxidative activity and the highest phenolic and flavonoid contents were detected in the methanol fraction, as a result of the contribution of not only phenols, but probably also lactones. The considerable antioxidative potential indicates possible applications in pharmacy and medicine.