Daphne mezereum L., an important medicinal plant in Scandinavian folk medicine, was used to treat ailments such as diarrhea, swelling and stomach pain. A range of natural compounds have been isolated, but little attention has been given to the polysaccharides in this plant. Previous work in our group have shown that a polysaccharide enriched fraction from the bark of D. mezereum exhibited pro-inflammatory effects. To pursue this further, the aim of the present work was to isolate and characterize these polysaccharides. From the ethanol-precipitate of a water extract, one neutral (DMP-NF) and one acidic (DMP-AF) fraction was isolated by anion-exchange chromatography. GC, GC-MS and 1D- and 2D-NMR were used to characterize the polysaccharide structures. DMP-NF appeared to be a mixture of arabinan, arabinogalactan and hemicelluloses such as xyloglucan, mannan and xylan. DMP-AF contained a pectic polysaccharide mainly consisting of an unusually long homogalacturonan backbone. Enzymatic treatment by pectinase of DMP-AF yielded DMP-ED, which contained a rhamnogalacturonan-I backbone with arabinan, galactan and arabinogalactan side chains. Both DMP-NF and DMP-ED induced IFN-γ and TNF-α secretion in peripheral blood mononuclear cells (PBMCs), DMP-ED being the most potent fraction. DMP-AF was less active, which might be due to a less sterically available rhamnogalacturonan-I domain.

With the advent of immunotherapies against cancers, autoimmune diseases and infections, there is a steady demand for novel medicines. New sources for discovery of potentially novel immunomodulatory compounds are therefore needed. Nature contains a large and diverse reservoir of novel compounds that can be exploited for their potential as new drugs, and exploring the pharmaceutical potential of medicinal plants used in traditional medicine is highly relevant. We aimed with this study to explore usage of medicinal plants in Scandinavian folk medicine against diseases interpreted to involve the immune system, and to further screen water extracts from previously overlooked medicinal plants in order to discover potential new sources of immunomodulatory compounds. We systematically investigated historical records dating back to the 1800s with an emphasis on plants used as treatment for wounds or diseases interpreted to be inflammatory. Of 74 candidate plants, 23 pharmacologically under-studied species were selected for further characterization. The plants were collected from their natural habitats in Southern Norway, air-dried, and subjected to boiling water and accelerated solvent extraction. The crude extracts were separated into polysaccharide-enriched fractions and C-18 solid phase extracted fractions. Immunological screenings were performed with all extracts and fractions. Monosaccharide composition and total phenolic content were determined and compared across all species. We identified 10 species with clear immune activating effects and 8 species with immune inhibitory effects by comparing cytokine production by human peripheral blood mononuclear cells, primary human T- and NK-cell proliferation, and nitric oxide production from macrophages. With this study, we provide a comprehensive overview of Scandinavian medicinal plants and their usage, and our findings support an approach of combining historical sources with modern pharmacology in the discovery of plant sources containing potentially new pharmacological compounds.

Daphne mezereum L. (Thymelaeaceae) was an important medicinal plant in Norway during the 18th and 19th centuries and used against diseases such as diarrhea, swelling, stomach pain, and tuberculosis. Five previously undescribed phenolic compounds, including two biflavonoids with a catechin core structure, two tricoumarins, and one bicoumarin, together with ten known compounds were isolated from a 50% EtOH extract of the bark of D. mezereum. Using NMR, HRESIMS, acid hydrolysis, and circular dichroism spectra, the biflavonoids were identified as 3′-hydroxygenkwanol A and 3′-hydroxydihydrodaphnodorin B, and the coumarins were identified as 3‴-O-acetyltriumbellin, triumbellin 4‴-O-β-d-glucopyranoside, and daphnogitin-7-O-β-d-glucopyranoside. The absolute configuration of dihydrodaphnodorin B was for the first time established as 2R, 3S, 2″S, 3″S. Daphnin, syringin, 3′-hydroxydihydrodaphnodorin B, dihydrodaphnodorin B, and neochamaejasmin A and B were identified as the major secondary metabolites in the extract. Neochamaejasmin A and B showed the most potent inhibition of TNF-α secretion in Con A stimulated peripheral blood mononuclear cells (PBMCs) with 71.3 ± 3.4 and 83.5 ± 11.5% inhibition, respectively, at 50 μM.

The plant kingdom serves as a valuable resource for cancer drug development. This study explored the antitumor activity of different sub-fractions (hexane, dichloromethane and methanol) of U. gallii (gorse) methanol extract in glioblastoma (U-87MG and U-373MG) and neuroblastoma (SH-SY5Y) cell lines, along with their phytochemical profiles. Cytotoxicity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, and cell cycle arrest and apoptosis were assessed through flow cytometry and by measuring reactive oxygen species (ROS) and protein expression levels. D7 and D8 dichloromethane sub-fractions significantly reduced cell viability, triggered early apoptosis in SH-SY5Y and U-87MG cells and specifically increased ROS levels in U-87MG cells. Western blot analyses showed that D7 increased p53, caspase-3, caspase-8 and γH2AX expression in SH-SY5Y and U-87MG cells, while D8 specifically elevated p53 in SH-SY5Y cells and caspase-3 in both cell lines. In U-373 cells, D7 and D8 markedly reduced cell viability, with D8 inducing necrosis. Morphological changes indicative of apoptosis were also observed in all cell lines. Bioinformatic analysis of UHPLC-MS and GC-MS data tentatively identified 20 metabolites in D7 and 15 in D8, primarily flavonoids. HPLC-DAD confirmed isoprunetin and genistein as the most abundant in D7 and D8, respectively, both isolated and identified by NMR spectroscopy. Most of the flavonoids identified have been reported as antitumor agents, suggesting that these compounds may be responsible for the observed pharmacological activity.