Comprehensive identification of polyphenolic metabolites in aspen knotwood by combination of 2D NMR and HPLC-HRMS.

INTRODUCTION: European aspen (Populus tremula L.) knotwood contains large amounts of polyphenolic metabolites, mainly flavonoids, and can be considered as a promising industrial-scale source of valuable bioactive compounds. Valorization of knotwood extractives requires detailed information on their chemical composition and a relevant analytical methodology. OBJECTIVE: This study proposes combined analytical strategy for non-targeted screening and identification of polyphenolic plant metabolites and is aimed at comprehensive characterization of knotwood extractives. MATERIALS AND METHODS: Aspen knotwood acetone extract with determined antioxidant activity was an object of the study. Two-dimensional NMR spectroscopy with Structure Elucidator expert system was used for preliminary search of major components and specific structures. Liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) with data-dependent MS/MS spectra acquisition was used as a complementary technique providing molecular-level characterization and identification of the detected metabolites. RESULTS: Twenty-eight phenolic metabolites were found and identified. Among them, flavonoids, aromadendrin and naringenin, as well as their glycosylated derivatives (mainly O-glucosides) and methyl ethers, dominated. Taxifolin and its 7-O-glucoside were detected as minor components. Other detected compounds are represented by p-coumaric acid and its rutinoside and small amounts of glycosylated ferulic acid. Nineteen of the detected compounds were discovered in aspen knotwood for the first time. The results were confirmed by preparative isolation of individual compounds and NMR studies. CONCLUSION: The proposed analytical strategy based on 2D NMR and HPLC-HRMS can be considered a powerful tool in the analysis of plant extractives and allowed for the identification and semi-quantification of a large number of polyphenols in aspen knotwood.

Polycommunin A, a dihydrocinnamoyl bibenzyl isolated from the moss Polýtrichum commúne, and its antioxidant activity.

A novel polyphenolic compound named Polycommunin A (1) was discovered in the aerial part of the common haircap moss (Polýtrichum commune) widely spread in boreal and temperate climate zones. Aqueous ethanol and extraction of the plant material with further isolation of polyphenolic fraction and preparative HPLC separation allowed obtaining individual compound and identifying it as dimeric dihydrocinnamoyl bibenzyl by NMR spectroscopy and high-resolution tandem mass spectrometry. Polycommunin A demonstrated high in vitro antioxidant activity determined by FRAP and PCL assays and comparable to that of Trolox and Quercetin.

Comprehensive Characterization of Secondary Metabolites in Fruits and Leaves of Cloudberry (Rubus chamaemorus L.).

Cloudberry (Rubus chamaemorus L.) is a circumpolar boreal plant rich in bioactive compounds and is widely used in food and in folk medicine. In this study, a combination of two-dimensional NMR spectroscopy and liquid chromatography-high-resolution mass spectrometry was used for the comprehensive characterization of secondary metabolites in cloudberry lipophilic and hydrophilic extracts. Special attention was paid to the leaf extractives, which are highly enriched in polyphenolic compounds, the content of which reaches 19% in the extract (in gallic acid equivalent). The chemical composition of the polyphenolic fraction is represented mainly by the glycosylated derivatives of flavonoids, hydroxycinnamic (primarily caffeic), gallic (including the structure of galloyl ascorbate) and ellagic acids, catechin, and procyanidins. The contents of aglycones in the polyphenolic fraction were 64 and 100 mg g-1 for flavonoids and hydroxycinnamic acids, respectively, while the content of free caffeic acid was 1.2 mg g-1. This determines the exceptionally high antioxidant activity of this fraction (750 mg g-1 in gallic acid equivalent) and the ability to scavenge superoxide anion radicals, which is 60% higher than that of Trolox. The lower polar fractions consist mainly of glycolipids, which include polyunsaturated linolenic acid (18:3), pentacyclic triterpenic acids, carotenoid lutein, and chlorophyll derivatives, among which pheophytin a dominates. Along with the availability, the high antioxidant and biological activities of cloudberry leaf extracts make them a promising source of food additives, cosmetics, and pharmaceuticals.

Comprehensive Characterization of Chemical Composition and Antioxidant Activity of Lignan-Rich Coniferous Knotwood Extractives.

A knotwood of coniferous trees containing large amounts of polyphenolic extractives is considered a promising industrial-scale source of lignans possessing antioxidant properties and other bioactivities. The present study is aimed at a detailed characterization of the chemical composition and antioxidant activity of lignan-rich extractives obtained from the knotwood of the Norway spruce, Scotch pine, Siberian fir, and Siberian larch growing in the European North of Russia as a region with a highly developed forest industry. To achieve this, a comprehensive approach based on a combination of two-dimensional NMR spectroscopy with high-performance liquid chromatography-high-resolution Orbitrap mass spectrometry, and the determination of antioxidant activity by the three complementary methods were proposed. The studied knotwood samples contained from 3.9 to 17% of extractive substances and were comparable to Trolox's antioxidant activity in the single-electron transfer processes and superoxide radical scavenging, which is associated with the predominance of polyphenolic compounds. The latter was represented by 12 tentatively identified monolignans and 27 oligolignans containing 3-5 phenylpropane units in their structure. The extracts were characterized by an identical set of lignans and differed only in the ratios of their individual compounds. Other components of the knotwood were flavonoids taxifolin, quercetin (Siberian larch), and three stilbenes (pinosylvin, its methyl ester, and pterostilbene), which were identified in the Scotch pine extractives. Sesquiterpene juvabione and its derivatives were found in extracts of Siberian larch knotwood.

New Oligomeric Dihydrochalcones in the Moss Polytrichum commune: Identification, Isolation, and Antioxidant Activity.

One of the most widespread representatives of mosses in the temperate and boreal latitudes of the Northern Hemisphere is common haircap (Polytrichum commune), which is known as the largest moss in the world and widely used in traditional herbal medicine. Polyphenolic compounds constitute one of the most important groups of biologically active secondary metabolites of P. commune, however, the available information on their chemical composition is still incomplete and contradictory. In the present study, a group of dihydrochalcone polyphenolic derivatives that were not previously found in mosses was isolated from P. commune biomass using pressurized liquid extraction with aqueous acetone. The combination of two-dimensional NMR spectroscopy and high-performance liquid chromatography-high-resolution mass spectrometry allowed for identifying them as 3-hydroxyphloretin oligomers formed through a carbon-carbon bond between phloroglucinol and pyrocatechol moieties ("head-to-tail" coupling), with a polymerization degree of 2-5. The individual compounds isolated by preparative reverse-phase HPLC had a purity of 71 to 97% and demonstrated high radical scavenging activity (17.5-42.5% with respect to Trolox) determined by the photochemiluminescence method. Along with the low toxicity predicted by QSAR/QSTR algorithms, this makes 3-hydroxyphloretin oligomers a promising source for the production of biologically active food additives and pharmaceuticals.