Ágnes Alberti

The aim of our study was the detailed polyphenol profiling of Juglans nigra and the characterization of the membrane permeability and antiproliferative properties of its main phenolics. A total of 161 compounds were tentatively identified in J. nigra bark, leaf, and pericarp extracts by ultrahigh-performance liquid chromatography–high-resolution tandem mass spectrometry (UHPLC-HR-MS/MS). Eight compounds including myricetin-3-O-rhamnoside (86), quercetin-3-O-rhamnoside (106), quercetin-3-O-xyloside (74), juglone (141), 1,2,3,4-tetrahydro-7,8-dihydroxy-4-oxonaphthalen-1-yl-6-O-galloyl-glucoside (92), ellagic acid (143), gallic acid (14), and ethyl gallate (58) were isolated from J. nigra pericarp. The in vitro antiproliferative activity of the isolated compounds was investigated against three human cancer cell lines, confirming that juglone (141) inhibits cell proliferation in all of them, and has similar activity as the clinical standards. The permeability of the isolated compounds across biological membranes was evaluated by the parallel artificial membrane permeability assay (PAMPA). Both juglone (141) and ethyl-gallate (58) showed positive results in the blood–brain-barrier-specific PAMPA-BBB study. Juglone (141) also possesses logPe values which indicates that it may be able to cross both the GI and BBB membranes via passive diffusion.

In this study, based on ethnobotanical data recorded in Transylvania, the polyphenolic compounds and the permeability of the aerial part’s extract of Tanacetum balsamita were investigated. Ultrahigh-performance liquid chromatography-tandem mass spectrometry was applied for the analysis of the extracts. Parallel artificial membrane permeability assay (PAMPA) for the gastrointestinal tract and the blood–brain barrier was conducted. In the ethanolic and aqueous extracts of the species traditionally used for wound, furuncle, and liver disorders, 92 polyphenols were characterized (e.g., flavonoid, hydroxycinnamic acid, catechin, dihydroxybenzoyl, lignan derivatives, and a monoterpene) including 54 compounds identified for the first time in the plant. In the PAMPA tests, eight components were shown to be capable of passive diffusion across the studied membranes. These include apigenin and seven methoxylated flavonoid derivatives. Based on these results, methoxylated flavonoids might promote the pharmacological potential of T. balsamita to be applied in the enhancement of novel remedies.

In this study, in vitro antioxidant, antimicrobial, cytotoxic, and cell migration effects of phenolic compounds of Lathyrus tuberosus leaves, known in the Transylvanian ethnomedicine, were investigated. Ultra-high-performance liquid chromatography-tandem mass spectrometry was employed for the analysis of the ethanolic and aqueous extracts. The antimicrobial properties were determined using a conventional microdilution technique. Total antioxidant capacity techniques were used using cell-free methods and cell-based investigations. Cytotoxic effects were conducted on 3T3 mouse fibroblasts and HaCaT human keratinocytes using a multiparametric method, assessing intracellular ATP, total nucleic acid, and protein levels. Cell migration was visualized by phase-contrast microscopy, employing conventional culture inserts to make cell-free areas. Together, 93 polyphenolic and monoterpenoid compounds were characterized, including flavonoid glycosides, lignans, hydroxycinnamic acid, and hydroxybenzoic acid derivatives, as well as iridoids and secoiridoids. The ethanolic extract showed high antioxidant capacity and strong antimicrobial activity against Bacillus subtilis (MIC₈₀ value: 354.37 ± 4.58 µg/mL) and Streptococcus pyogenes (MIC₈₀ value: 488.89 ± 4.75 µg/mL). The abundance of phenolic compounds and the results of biological tests indicate the potential for L. tuberosus to serve as reservoirs of bioactive compounds and to be used in the development of novel nutraceuticals.

Four cyclic diarylheptanoids—carpinontriols A (1) and B (2), giffonin X (3) and 3,12,17-trihydroxytricyclo [12.3.1.1^2,6]nonadeca-1(18),2(19),3,5,14,16-hexaene-8,11-dione (4)—were isolated from Carpinus betulus (Betulaceae). Chemical stability of the isolated diarylheptanoids was evaluated as a function of storage temperature (−15, 5, 22 °C) and time (12 and 23 weeks). The effect of the solvent and the pH (1.2, 6.8, 7.4) on the stability of these diarylheptanoids was also investigated. Compounds 2 and 4 showed good stability both in aqueous and methanolic solutions at all investigated temperatures. Only 2 was stable at all three studied biorelevant pH values. Degradation products of 1 and 3 were formed by the elimination of a water molecule from the parent compounds, as confirmed by ultrahigh-performance liquid chromatography–high-resolution tandem mass spectrometry (UHPLC-HR-MS). The permeability of the compounds across biological membranes was evaluated by the parallel artificial membrane permeability assay (PAMPA). Compound 3 possesses a logPe value of −5.92 ± 0.04 in the blood–brain barrier-specific PAMPA-BBB study, indicating that it may be able to cross the blood–brain barrier via passive diffusion. The in vitro antiproliferative activity of the compounds was investigated against five human cancer cell lines, confirming that 1 inhibits cell proliferation in A2058 human metastatic melanoma cells.

Seven diarylheptanoids were isolated from Corylus maxima by flash chromatography and semipreparative high-performance liquid chromatography (HPLC) and identified by Orbitrap^® mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy as linear diarylheptanoids: hirsutanonol-5-O-β-D-glucopyranoside (1), platyphyllonol-5-O-β-D-xylopyranoside (4), platyphyllenone (5); and cyclic derivatives: alnusonol-11-O-β-D-glucopyranoside (6), alnusone (7), giffonin F (8), carpinontriol B (9). Cyclic diarylheptanoids are reported in C. maxima for the first time. The aqueous stability of the isolated compounds and other characteristic constituents of C. maxima, oregonin (2), hirsutenone (3), quercitrin (10) and myricitrin (11) was evaluated at pH 1.2, 6.8 and 7.4. The passive diffusion of the constituents across biological membranes was investigated by parallel artificial membrane permeability assay for the gastrointestinal tract (PAMPA-GI) and the blood–brain barrier (PAMPA-BBB) methods. The cyclic diarylheptanoid aglycones and quercitrin were stable at all investigated pH values, while a pH-dependent degradation of the other compounds was observed. A validated ultrahigh-performance liquid chromatography-diode-array detection (UHPLC-DAD) method was utilized for the determination of compound concentrations. The structures of the degradation products were characterized by UHPLC-Orbitrap^® MS. Platyphyllenone and alnusone possessed log P_e values greater than −5.0 and −6.0 in the PAMPA-GI and PAMPA-BBB studies, respectively, indicating their ability to cross the membranes via passive diffusion. However, only alnusone can be considered to have both good aqueous stability and satisfactory membrane penetration ability.

Medicinal plants are widely used in folk medicine but quite often their composition and biological effects are hardly known. Our study aimed to analyze the composition, cytotoxicity, antimicrobial, antioxidant activity and cellular migration effects of Anthyllis vulneraria, Fuchsia magellanica, Fuchsia triphylla and Lysimachia nummularia used in the Romanian ethnomedicine for wounds. Liquid chromatography with mass spectrometry (LC-MS/MS) was used to analyze 50% (v/v) ethanolic and aqueous extracts of the plants’ leaves. Antimicrobial activities were estimated with a standard microdilution method. The antioxidant properties were evaluated by validated chemical cell-free and biological cell-based assays. Cytotoxic effects were performed on mouse fibroblasts and human keratinocytes with a plate reader-based method assessing intracellular adenosine triphosphate (ATP), nucleic acid and protein contents and also by a flow cytometer-based assay detecting apoptotic–necrotic cell populations. Cell migration to cover cell-free areas was visualized by time-lapse phase-contrast microscopy using standard culture inserts. Fuchsia species showed the strongest cytotoxicity and the highest antioxidant and antimicrobial activity. However, their ethanolic extracts facilitated cell migration, most probably due to their various phenolic acid, flavonoid and anthocyanin derivatives. Our data might serve as a basis for further animal experiments to explore the complex action of Fuchsia species in wound healing assays.

Horseradish hairy root cultures are suitable plant tissue organs to study the glucosinolate–myrosinase–isothiocyanate system and also to produce the biologically active isothiocyanates and horseradish peroxidase, widely used in molecular biology. Fifty hairy root clones were isolated after Agrobacterium rhizogenes infection of surface sterilized Armoracia rusticana petioles and leaf blades, from which 21 were viable after antibiotic treatment. Biomass properties (e.g., dry weight %, daily growth index), glucosinolate content (analyzed by liquid chromatography-electronspray ionization-mass spectrometry (LC-ESI-MS/MS)), isothiocyanate and nitrile content (analyzed by gas chromatography-mass spectrometry (GC-MS)), myrosinase (on-gel detection) and horseradish peroxidase enzyme patterns (on-gel detection and spectrophotometry), and morphological features were examined with multi-variable statistical analysis. In addition to the several positive and negative correlations, the most outstanding phenomenon was many parameters of the hairy root clones showed dependence on the organ of origin. Among others, the daily growth index, sinigrin, glucobrassicin, 3-phenylpropionitrile, indole-3-acetonitrile and horseradish peroxidase values showed significantly higher levels in horseradish hairy root cultures initiated from leaf blades.