Chloride, glutathiones, and insect-derived elicitors introduced into the xylem trigger electrical signaling.

Ricca assays allow the direct introduction of compounds extracted from plants or the organisms that attack them into the leaf vasculature. Using chromatographic fractionation of Arabidopsis (Arabidopsis thaliana) leaf extracts, we found glutamate was the most active low mass elicitor of membrane depolarization. However, other known elicitors of membrane depolarization are generated in the wound response. These include unstable aglycones generated by glucosinolate (GSL) breakdown. None of the aglycone-derived GSL-breakdown products, including nitriles and isothiocyanates, that we tested using Ricca assays triggered electrical activity. Instead, we found that glutathione and the GSL-derived compound sulforaphane glutathione triggered membrane depolarizations. These findings identify a potential link between GSL breakdown and glutathione in the generation of membrane depolarizing signals. Noting that the chromatographic fractionation of plant extracts can dilute or exchange ions, we found that Cl- caused glutamate receptor-like3.3-dependent membrane depolarizations. In summary, we show that, in addition to glutamate, glutathione derivatives as well as chloride ions will need to be considered as potential elicitors of wound-response membrane potential change. Finally, by introducing aphid (Brevicoryne brassicae) extracts or the flagellin-derived peptide flg22 into the leaf vasculature we extend the use of Ricca assays for the exploration of insect/plant and bacteria/plant interactions.

Spatial and evolutionary predictability of phytochemical diversity.

To cope with environmental challenges, plants produce a wide diversity of phytochemicals, which are also the source of numerous medicines. Despite decades of research in chemical ecology, we still lack an understanding of the organization of plant chemical diversity across species and ecosystems. To address this challenge, we hypothesized that molecular diversity is not only related to species diversity, but also constrained by trophic, climatic, and topographical factors. We screened the metabolome of 416 vascular plant species encompassing the entire alpine elevation range and four alpine bioclimatic regions in order to characterize their phytochemical diversity. We show that by coupling phylogenetic information, topographic, edaphic, and climatic variables, we predict phytochemical diversity, and its inherent composition, of plant communities throughout landscape. Spatial mapping of phytochemical diversity further revealed that plant assemblages found in low to midelevation habitats, with more alkaline soils, possessed greater phytochemical diversity, whereas alpine habitats possessed higher phytochemical endemism. Altogether, we present a general tool that can be used for predicting hotspots of phytochemical diversity in the landscape, independently of plant species taxonomic identity. Such an approach offers promising perspectives in both drug discovery programs and conservation efforts worldwide.

Antiprotozoal Activity of Plants Used in the Management of Sleeping Sickness in Angola and Bioactivity-Guided Fractionation of Brasenia schreberi J.F.Gmel and Nymphaea lotus L. Active against T. b. rhodesiense.

Folk medicine is widely used in Angola, even for human African trypanosomiasis (sleeping sickness) in spite of the fact that the reference treatment is available for free. Aiming to validate herbal remedies in use, we selected nine medicinal plants and assessed their antitrypanosomal activity. A total of 122 extracts were prepared using different plant parts and solvents. A total of 15 extracts from seven different plants exhibited in vitro activity (>70% at 20 µg/mL) against Trypanosoma brucei rhodesiense bloodstream forms. The dichloromethane extract of Nymphaea lotus (leaves and leaflets) and the ethanolic extract of Brasenia schreberi (leaves) had IC50 values ≤ 10 µg/mL. These two aquatic plants are of particular interest. They are being co-applied in the form of a decoction of leaves because they are considered by local healers as male and female of the same species, the ethnotaxon "longa dia simbi". Bioassay-guided fractionation led to the identification of eight active molecules: gallic acid (IC50 0.5 µg/mL), methyl gallate (IC50 1.1 µg/mL), 2,3,4,6-tetragalloyl-glucopyranoside, ethyl gallate (IC50 0.5 µg/mL), 1,2,3,4,6-pentagalloyl-ß-glucopyranoside (IC50 20 µg/mL), gossypetin-7-O-ß-glucopyranoside (IC50 5.5 µg/mL), and hypolaetin-7-O-glucoside (IC50 5.7 µg/mL) in B. schreberi, and 5-[(8Z,11Z,14Z)-heptadeca-8,11,14-trienyl] resorcinol (IC50 5.3 µg/mL) not described to date in N. lotus. Five of these active constituents were detected in the traditional preparation. This work provides the first evidence for the ethnomedicinal use of these plants in the management of sleeping sickness in Angola.

Wound-response jasmonate dynamics in the primary vasculature.

The links between wound-response electrical signalling and the activation of jasmonate synthesis are unknown. We investigated damage-response remodelling of jasmonate precursor pools in the Arabidopsis thaliana leaf vasculature. Galactolipids and jasmonate precursors in primary veins from undamaged and wounded plants were analysed using MS-based metabolomics and NMR. In parallel, DAD1-LIKE LIPASEs (DALLs), which control the levels of jasmonate precursors in veins, were identified. A novel galactolipid containing the jasmonate precursor 12-oxo-phytodienoic acid (OPDA) was identified in veins: sn-2-O-(cis-12-oxo-phytodienoyl)-sn-3-O-(ß-galactopyranosyl) glyceride (sn-2-OPDA-MGMG). Lower levels of sn-1-OPDA-MGMG were also detected. Vascular OPDA-MGMGs, sn-2-18:3-MGMG and free OPDA pools were reduced rapidly in response to damage-activated electrical signals. Reduced function dall2 mutants failed to build resting vascular sn-2-OPDA-MGMG and OPDA pools and, upon wounding, dall2 produced less jasmonoyl-isoleucine (JA-Ile) than the wild-type. DALL3 acted to suppress excess JA-Ile production after wounding, whereas dall2 dall3 double mutants strongly reduce jasmonate signalling in leaves distal to wounds. LOX6 and DALL2 function to produce OPDA and the non-bilayer-forming lipid sn-2-OPDA-MGMG in the primary vasculature. Membrane depolarizations trigger rapid depletion of these molecules. We suggest that electrical signal-dependent lipid phase changes help to initiate vascular jasmonate synthesis in wounded leaves.

Highlighting the Phototherapeutical Potential of Fungal Pigments in Various Fruiting Body Extracts with Informed Feature-Based Molecular Networking.

Fungal pigments are characterized by a diverse set of chemical backbones, some of which present photosensitizer-like structures. From the genus Cortinarius, for example, several biologically active photosensitizers have been identified leading to the hypothesis that photoactivity might be a more general phenomenon in the kingdom Fungi. This paper aims at testing the hypothesis. Forty-eight fruiting body-forming species producing pigments from all four major biosynthetic pathways (i.e., shikimate-chorismate, acetate-malonate, mevalonate, and nitrogen heterocycles) were selected and submitted to a workflow combining in vitro chemical and biological experiments with state-of-the-art metabolomics. Fungal extracts were profiled by high-resolution mass spectrometry and subsequently explored by spectral organization through feature-based molecular networking (FBMN), including advanced metabolite dereplication techniques. Additionally, the photochemical properties (i.e., light-dependent production of singlet oxygen), the phenolic content, and the (photo)cytotoxic activity of the extracts were studied. Different levels of photoactivity were found in species from all four metabolic groups, indicating that light-dependent effects are common among fungal pigments. In particular, extracts containing pigments from the acetate-malonate pathway, e.g., extracts from Bulgaria inquinans, Daldinia concentrica, and Cortinarius spp., were not only efficient producers of singlet oxygen but also exhibited photocytotoxicity against three different cancer cell lines. This study explores the distribution of photobiological traits in fruiting body forming fungi and highlights new sources for phototherapeutics.

Evaluation of the Antiseizure Activity of Endemic Plant Halfordia kendack Guillaumin and Its Main Constituent, Halfordin, on a Zebrafish Pentylenetetrazole (PTZ)-Induced Seizure Model.

Epilepsy is a neurological disease that burdens over 50 million people worldwide. Despite the considerable number of available antiseizure medications, it is estimated that around 30% of patients still do not respond to available treatment. Herbal medicines represent a promising source of new antiseizure drugs. This study aimed to identify new drug lead candidates with antiseizure activity from endemic plants of New Caledonia. The crude methanolic leaf extract of Halfordia kendack Guillaumin (Rutaceae) significantly decreased (75 µg/mL and 100 µg/mL) seizure-like behaviour compared to sodium valproate in a zebrafish pentylenetetrazole (PTZ)-induced acute seizure model. The main coumarin compound, halfordin, was subsequently isolated by liquid-liquid chromatography and subjected to locomotor, local field potential (LFP), and gene expression assays. Halfordin (20 µM) significantly decreased convulsive-like behaviour in the locomotor and LFP analysis (by 41.4% and 60%, respectively) and significantly modulated galn, and penka gene expression.

Future directions for the discovery of natural product-derived immunomodulating drugs: an IUPHAR positional review.

Drug discovery from natural sources is going through a renaissance, having spent many decades in the shadow of synthetic molecule drug discovery, despite the fact that natural product-derived compounds occupy a much greater chemical space than those created through synthetic chemistry methods. With this new era comes new possibilities, not least the novel targets that have emerged in recent times and the development of state-of-the-art technologies that can be applied to drug discovery from natural sources. Although progress has been made with some immunomodulating drugs, there remains a pressing need for new agents that can be used to treat the wide variety of conditions that arise from disruption, or over-activation, of the immune system; natural products may therefore be key in filling this gap. Recognising that, at present, there is no authoritative article that details the current state-of-the-art of the immunomodulatory activity of natural products, this in-depth review has arisen from a joint effort between the International Union of Basic and Clinical Pharmacology (IUPHAR) Natural Products and Immunopharmacology Sections, with contributions from a number of world-leading researchers in the field of natural product drug discovery, to provide a "position statement" on what natural products has to offer in the search for new immunomodulatory argents. To this end, we provide a historical look at previous discoveries of naturally occurring immunomodulators, present a picture of the current status of the field and provide insight into the future opportunities and challenges for the discovery of new drugs to treat immune-related diseases.

Combining Activity Profiling with Advanced Annotation to Accelerate the Discovery of Natural Products Targeting Oncogenic Signaling in Melanoma.

The discovery of bioactive natural products remains a time-consuming and challenging task. The ability to link high-confidence metabolite annotations in crude extracts with activity would be highly beneficial to the drug discovery process. To address this challenge, HPLC-based activity profiling and advanced UHPLC-HRMS/MS metabolite profiling for annotation were combined to leverage the information obtained from both approaches on a crude extract scaled down to the submilligram level. This strategy was applied to a subset of an extract library screening aiming to identify natural products inhibiting oncogenic signaling in melanoma. Advanced annotation and data organization enabled the identification of compounds that were likely responsible for the activity in the extracts. These compounds belonged to two different natural product scaffolds, namely, brevipolides from a Hyptis brevipes extract and methoxylated flavonoids identified in three different extracts of Hyptis and Artemisia spp. Targeted isolation of these prioritized compounds led to five brevipolides and seven methoxylated flavonoids. Brevipolide A (1) and 6-methoxytricin (9) were the most potent compounds from each chemical class and displayed AKT activity inhibition with an IC50 of 17.6 ± 1.6 and 4.9 ± 0.2 µM, respectively.

Targeted Isolation of Antibiotic Brominated Alkaloids from the Marine Sponge Pseudoceratina durissima Using Virtual Screening and Molecular Networking.

Many targeted natural product isolation approaches rely on the use of pre-existing bioactivity information to inform the strategy used for the isolation of new bioactive compounds. Bioactivity information can be available either in the form of prior assay data or via Structure Activity Relationship (SAR) information which can indicate a potential chemotype that exhibits a desired bioactivity. The work described herein utilizes a unique method of targeted isolation using structure-based virtual screening to identify potential antibacterial compounds active against MRSA within the marine sponge order Verongiida. This is coupled with molecular networking-guided, targeted isolation to provide a novel drug discovery procedure. A total of 12 previously reported bromotyrosine-derived alkaloids were isolated from the marine sponge species Pseudoceratina durissima, and the compound, (+)-aeroplysinin-1 (1) displayed activity against the MRSA pathogen (MIC: <32 µg/mL). The compounds (1−3, 6 and 9) were assessed for their central nervous system (CNS) interaction and behavioral toxicity to zebrafish (Danio rerio) larvae, whereby several of the compounds were shown to induce significant hyperactivity. Anthelmintic activity against the parasitic nematode Haemonchus contorutus was also evaluated (2−4, 6−8).

Correlation of chemical profiles obtained from 1 H-NMR and LC-MS metabolomics with α-glucosidase inhibition activity for varietal selections of Ficus deltoidea.

INTRODUCTION: Ficus deltoidea Jack (Moraceae) is a plant used in Malaysia to treat various ailments, including diabetes. The presence of several varieties raises essential questions regarding which is the potential bioactive variety and what are the bioactive metabolites. OBJECTIVES: Here, we explored the phytochemical diversity of the seven varieties from Peninsular Malaysia using Nuclear Magnetic Resonance (NMR) and Liquid Chromatography-Mass Spectrometry (LC-MS) analyses and correlated it with the α-glucosidase inhibitory activity. METHODOLOGY: The Nuclear Overhauser Effect Spectroscopy (NOESY) One-Dimensional (1D)-NMR and LC-MS data were processed, annotated, and correlated with in vitro α-glucosidase inhibitory using multivariate data analysis. RESULTS: The α-glucosidase results demonstrated that different varieties have varying inhibitory effects, with the highest inhibition rate being F. deltoidea var. trengganuensis and var. kunstleri. Furthermore, diverse habitats and plant ages could also influence the inhibitory rate. The heat map from NMR and LC-MS profiles showed unique patterns according to varying levels of α-glucosidase inhibition rate. The Partial Least Squares (PLS) model constructed from both NMR and LC-MS further confirmed the correlation between the α-glucosidase inhibition rate of F. deltoidea varieties and its metabolite profiles. The Variable Influence on Projection (VIP) and correlation coefficient (p(corr)) values values were used to determine the highly relevant metabolites for explaining the anticipated inhibitory action. CONCLUSION: NMR and LC-MS annotations allow the identification of flavan-3-ols and proanthocyanidins as the key bioactive factors. Our current results demonstrated the value of multivariate data analysis to predict the quality of herbal materials from both biological and chemical aspects.

Metabolomics in Ecology and Bioactive Natural Products Discovery: Challenges and Prospects for a Comprehensive Study of the Specialised Metabolome.

Metabolomics is playing an increasingly prominent role in chemical ecology and in the discovery of bioactive natural products (NPs). The identification of metabolites is a common/central objective in both research fields. NPs have significant biological properties and play roles in multiple chemical-ecological interactions. Classically, in pharmacognosy, their chemical structure is determined after a complex process of isolating and interpreting spectroscopic data. With the advent of powerful analytical techniques such as liquid chromatography-mass spectrometry (LC-MS) the annotation process of the specialised metabolome of plants and microorganisms has improved considerably. In this article, we summarise the possibilities opened by these advances and illustrate how we harnessed them in our own research to automate annotations of NPs and target the isolation of key compounds. In addition, we are also discussing the analytical and computational challenges associated with these emerging approaches and their perspective.

Molecular and Functional Analysis of Sunitinib-Resistance Induction in Human Renal Cell Carcinoma Cells.

Resistance in clear cell renal cell carcinoma (ccRCC) against sunitinib is a multifaceted process encompassing numerous molecular aberrations. This induces clinical complications, reducing the treatment success. Understanding these aberrations helps us to select an adapted treatment strategy that surpasses resistance mechanisms, reverting the treatment insensitivity. In this regard, we investigated the dominant mechanisms of resistance to sunitinib and validated an optimized multidrug combination to overcome this resistance. Human ccRCC cells were exposed to single or chronic treatment with sunitinib to obtain three resistant clones. Upon manifestation of sunitinib resistance, morphometric changes in the cells were observed. At the molecular level, the production of cell membrane and extracellular matrix components, chemotaxis, and cell cycle progression were dysregulated. Molecules enforcing the cell cycle progression, i.e., cyclin A, B1, and E, were upregulated. Mass spectrometry analysis revealed the intra- and extracellular presence of N-desethyl sunitinib, the active metabolite. Lysosomal sequestration of sunitinib was confirmed. After treatment with a synergistic optimized drug combination, the cell metabolic activity in Caki-1-sunitinib-resistant cells and 3D heterotypic co-cultures was reduced by >80%, remaining inactive in non-cancerous cells. These results demonstrate geno- and phenotypic changes in response to sunitinib treatment upon resistance induction. Mimicking resistance in the laboratory served as a platform to study drug responses.

Coumarins from Seseli devenyense Simonk.: Isolation by Liquid-Liquid Chromatography and Potential Anxiolytic Activity Using an In Vivo Zebrafish Larvae Model.

Different types of anxiety disorders have become the number one mental health issue in developed countries. The search for new, safer and effective drug-like molecules among naturally derived substances faces two difficulties: an efficient method of isolation compounds with a high-purity and high-throughput animal model for activity assay. Thus, the aim of the present study was to isolate by liquid-liquid chromatography high-purity rare coumarins from the fruits of Seseli devenyense Simonk. and evaluate their anxiolytic effect (defined as reversed thimotaxis) using a 5-days post-fertilization (dpf) Danio rerio larvae model. Liquid-liquid chromatography enabled the isolation of one simple hydroxycoumarin (devenyol) and four pyranocoumarins (cis-khellactone, d-laserpitin, isolaserpitin and octanoyllomatin). The anxiolytic effect was defined as a decrease in the time spent in the boundaries of the living space (also described as reversed thigmotaxis). Our results show that all isolated courmarins exerted a significant influence on the anxiety behavior (anxiolytic activity) in the zebrafish larvae model. According to our knowledge, this is the first report of anxiolytic activity of pyranocoumarins and devenyol.

Liquid-Liquid Chromatography Separation of Guaiane-Type Sesquiterpene Lactones from Ferula penninervis Regel & Schmalh. and Evaluation of Their In Vitro Cytotoxic and Melanin Inhibitory Potential.

Ferula penninervis Regel & Schmalh. is a perennial plant used in Kazakh traditional folk medicine to treat epilepsy, neurosis, rheumatism, gastroduodenal ulcers, dyspepsia, wounds, abscesses or tumors. The aim of this work was to isolate series of sesquiterpene lactones from a crude methanolic root extract and investigate their in vitro cytotoxic potential against androgen-dependent prostate cancer LNCaP and epithelial prostate PNT2 cells, as well as to evaluate their melanin production inhibitory effects in murine melanoma B16F10 cells stimulated with α-melanocyte-stimulating hormone (αMSH). Two new (penninervin P and penninervin Q) and five known (olgin, laferin, olgoferin, oferin and daucoguainolactone F) guaiane-type sesquiterpene lactones were isolated with the use of a simple and fast liquid-liquid chromatography method. Olgin and laferin showed the most promising cytotoxic effects in LNCaP cells (IC50 of 31.03 and 23.26 µg/mL, respectively). Additionally, olgin, laferin, olgoferin, and oferin (10 µg/mL) potently impaired melanin release (40.67-65.48% of αMSH + cells) without influencing the viability of B16F10 cells. In summary, our findings might indicate that guaiane-type sesquiterpene lactones from F. penninervis could be regarded as promising candidates for further research in discovering new therapeutic agents with anti-prostate cancer and skin depigmentation properties.

Surface sensing triggers a broad-spectrum antimicrobial response in Pseudomonas aeruginosa.

Interspecies bacterial competition may occur via cell-associated or secreted determinants and is key to successful niche colonization. We previously evolved Pseudomonas aeruginosa in the presence of Staphylococcus aureus and identified mutations in the Wsp surface-sensing signalling system. Surprisingly, a ΔwspF mutant, characterized by increased c-di-GMP levels and biofilm formation capacity, showed potent killing activity towards S. aureus in its culture supernatant. Here, we used an unbiased metabolomic analysis of culture supernatants to identify rhamnolipids, alkyl quinoline N-oxides and two siderophores as members of four chemical clusters, which were more abundant in the ΔwspF mutant supernatants. Killing activities were quorum-sensing controlled but independent of c-di-GMP levels. Based on the metabolomic analysis, we formulated a synthetic cocktail of four compounds, showing broad-spectrum anti-bacterial killing, including both Gram-positive and Gram-negative bacteria. The combination of quorum-sensing-controlled killing and Wsp-system mediated biofilm formation endows P. aeruginosa with capacities essential for niche establishment and host colonization.

Wound- and mechanostimulated electrical signals control hormone responses.

Plants in nature are constantly exposed to organisms that touch them and wound them. A highly conserved response to these stimuli is a rapid collapse of membrane potential (i.e. a decrease of electrical field strength across membranes). This can be coupled to the production and/or action of jasmonate or ethylene. Here, the various types of electrical signals in plants are discussed in the context of hormone responses. Genetic approaches are revealing genes involved in wound-induced electrical signalling. These include clade 3 GLUTAMATE RECEPTOR-LIKE (GLR) genes, Arabidopsis H+ -ATPases (AHAs), RESPIRATORY BURST OXIDASE HOMOLOGUEs (RBOHs), and genes that determine cell wall properties. We briefly review touch- and wound-induced increases in cytosolic Ca2+ concentrations and their temporal relationship to electrical activities. We then look at the questions that need addressing to link mechanostimulation and wound-induced electrical activity to hormone responses. Utilizing recently published results, we also present a hypothesis for wound-response leaf-to-leaf electrical signalling. This model is based on rapid electro-osmotic coupling between the phloem and xylem. The model suggests that the depolarization of membranes within the vascular matrix triggered by physical stimuli and/or chemical elicitors is linked to changes in phloem turgor and that this plays vital roles in leaf-to-leaf electrical signal propagation.

Paecilosetin Derivatives as Potent Antimicrobial Agents from Isaria farinosa.

Fifty-seven entomopathogenic microorganisms were screened against human pathogens and subjected to mass spectrometry molecular networking based dereplication. Isaria farinosa BSNB-1250, shown to produce potentially novel biologically active metabolites, was grown on a large scale on potato dextrose agar, and paecilosetin (1) and five new analogues (2-6) were subsequently isolated. The structures of the new compounds were elucidated using 1D and 2D NMR. The absolute configurations of compounds 1-6 were determined using Mosher ester derivatives (1, 3, 4), comparison of experimental and calculated ECD spectra (2-4 and 6), and single-crystal X-ray diffraction analysis (5). Compounds 1 and 5 exhibited strong antibacterial activity against MSSA and MRSA with MIC values of 1-2 µg/mL.

Generation of Stilbene Antimicrobials against Multiresistant Strains of Staphylococcus aureus through Biotransformation by the Enzymatic Secretome of Botrytis cinerea.

The biotransformation of a mixture of resveratrol and pterostilbene was performed by the protein secretome of Botrytis cinerea. Several reaction conditions were tested to overcome solubility issues and to improve enzymatic activity. Using MeOH as cosolvent, a series of unusual methoxylated compounds was generated. The reaction was scaled-up, and the resulting mixture purified by semipreparative HPLC-PDA-ELSD-MS. Using this approach, 15 analogues were isolated in one step. Upon full characterization by NMR and HRMS analyses, eight of the compounds were new. The antibacterial activities of the isolated compounds were evaluated in vitro against the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The selectivity index was calculated based on cytotoxic assays performed against human liver carcinoma cells (HepG2) and the human breast epithelial cell line (MCF10A). Some compounds revealed remarkable antibacterial activity against multidrug-resistant strains of S. aureus with moderate human cell line cytotoxicity.

Production of Highly Active Antiparasitic Compounds from the Controlled Halogenation of the Arrabidaea brachypoda Crude Plant Extract.

Direct halogenation of phenolic compounds present in the CH2Cl2 extract of the roots of Arrabidaea brachypoda was investigated to enhance chemodiversity. The approach is based on eco-friendly reactions using NaBr, NaI, and NaCl in aqueous media to generate multiple "unnatural" halogenated natural products from crude extracts. The halogenation reactions, monitored by UHPLC-PDA-ELSD-MS, were optimized to generate mono-, di-, or trihalogenated derivatives. To isolate these compounds, the reactions were scaled up and the halogenated analogues were isolated by semipreparative HPLC-UV and fully characterized by NMR and HR-MS data. All of the original 16 halogenated derivatives were evaluated for their antiparasitic activities against the parasites Leishmania amazonensis and Trypanosoma cruzi. Compounds presenting selective antiparasitic activities against one or both parasites with IC50 values comparable to the reference were identified.

Phytochemical and Biological Investigation of Helianthemum nummularium, a High-Altitude Growing Alpine Plant Overrepresented in Ungulates Diets.

Helianthemum nummularium is a European shrub growing at high altitude where it copes with a high level of stress. It was found to be overexpressed in ungulates diets compared to more abundant surrounding plants. These elements combined with the fact that H. nummularium from the Alps has never been investigated prompted us to study the phytochemical composition of its aerial parts. The analysis of the polar extract allowed for the isolation of eight compounds: p-hydroxybenzoic acid, tiliroside, kaempferol, astragalin, quercetin, plantainoside B, quercetin-3-O-glucoside, and quercetin-3-O-glucuronide. We investigated the effect of the polar extract and isolated compounds on nuclear factor erythroid 2-related factor 2 transcription factor, which regulates the expression of a wide variety of cytoprotective genes. We found that the ethanolic extract activates the expression of nuclear factor erythroid 2-related factor 2 in a dose-dependent manner, whereas the pure compounds were much less active. The activation of the nuclear factor erythroid 2-related factor 2 pathway by the plant extract could pave the way for studies to promote healthy aging through protection of cells against oxidative stress. Moreover, the isolated compounds could be investigated alone or in combination in the perspective of making the link between the ungulate's preference for this plant and possible use of it for self-medication.