The usefulness of anti-inflammatory drugs as an adjunct therapy to improve outcomes in COVID-19 patients is intensely discussed in this paper. Willow bark (Salix cortex) has been used for centuries to relieve pain, inflammation, and fever. Its main active ingredient, salicin, is metabolized in the human body into salicylic acid, the precursor of the commonly used pain drug acetylsalicylic acid (ASA). Here, we report on the in vitro anti-inflammatory efficacy of two methanolic Salix extracts, standardized to phenolic compounds, in comparison to ASA in the context of a SARS-CoV-2 peptide challenge. Using SARS-CoV-2 peptide/IL-1β- or LPS-activated human PBMCs and an inflammatory intestinal Caco-2/HT29-MTX co-culture, Salix extracts, and ASA concentration-dependently suppressed prostaglandin E2 (PGE2), a principal mediator of inflammation. The inhibition of COX-2 enzyme activity, but not protein expression was observed for ASA and one Salix extract. In activated PBMCs, the suppression of relevant cytokines (i.e., IL-6, IL-1β, and IL-10) was seen for both Salix extracts. The anti-inflammatory capacity of Salix extracts was still retained after transepithelial passage and liver cell metabolism in an advanced co-culture model system consisting of intestinal Caco-2/HT29-MTX cells and differentiated hepatocyte-like HepaRG cells. Taken together, our in vitro data suggest that Salix extracts might present an additional anti-inflammatory treatment option in the context of SARS-CoV-2 peptides challenge; however, more confirmatory data are needed.

Salix cortex-containing medicine is used against pain conditions, fever, headaches, and inflammation, which are partly mediated via arachidonic acid-derived prostaglandins (PGs). We used an activity-guided fractionation strategy, followed by structure elucidation experiments using LC-MS/MS, CD-spectroscopy, and 1D/2D NMR techniques, to identify the compounds relevant for the inhibition of PGE2 release from activated human peripheral blood mononuclear cells. Subsequent compound purification by means of preparative and semipreparative HPLC revealed 2′-O-acetylsalicortin (1), 3′-O-acetylsalicortin (2), 2′-O-acetylsalicin (3), 2′,6′-O-diacetylsalicortin (4), lasiandrin (5), tremulacin (6), and cinnamrutinose A (7). In contrast to 3 and 7, compounds 1, 2, 4, 5, and 6 showed inhibitory activity against PGE2 release with different potencies. Polyphenols were not relevant for the bioactivity of the Salix extract but salicylates, which degrade to, e.g., catechol, salicylic acid, salicin, and/or 1-hydroxy-6-oxo-2-cycohexenecarboxylate. Inflammation presents an important therapeutic target for pharmacological interventions; thus, the identification of relevant key drugs in Salix could provide new prospects for the improvement and standardization of existing clinical medicine.

Willow bark is traditionally used for pharmaceutical purposes. Evaluation is so far based on the salicylate content, however, health promoting effects of extracts might be attributed to the interaction of those salicylates with other compounds, which support and complement their action. So far, only S. purpurea, S. daphnoides, and S. fragilis are included in pharmaceutical extracts. Crossing with other species could result in a more diverse secondary metabolite profile with higher pharmacological value. With the help of targeted inter- and intraspecific crossing, new chemotypes were generated, whereby nine different Salix genotypes (S. alba, S. daphnoides, S. humboldtiana, S. lasiandra, S. nigra, S. pentandra, S. purpurea, S. x rubens, S. viminalis) were included in the study. Based on substances known for their health promoting potential and characteristic for Salix (selected phenolic compounds including salicylates), a targeted metabolomics analysis and clustering of 92 generated Salix clones was performed revealing four different cluster/chemoprofiles. In more specific, one group is formed by S. daphnoides clones and inter- and intraspecific hybrids, a second group by S. viminalis clones and inter- and intraspecific hybrids, a third group generally formed by S. alba, S. pentandra, S. x rubens, and S. lasiandra clones and hybrids, and a fourth group by S. purpurea clones and inter- and intraspecific hybrids. Clustering on the basis of the selected phenolic compounds can be used for identifying Salix clones with a different compound profile. New combinations of secondary plant metabolites offer the chance to identify Salix crosses with improved effects on human health.

Willow (Salix L.) trees can be used in the field of sustainable solutions, for instance, they contribute to the protection against water, soil and wind erosion. On the other hand, short-rotation plantation of the fastgrowing willow trees can help generate renewable energy by using the heartwood for biomass production, like fuel or fibre. Additionally, willow bark is an important source for the plant-based drug production. The willows have been used as phytopharmaceuticals against inflammatory rheumatic disease since ancient times. Nowadays, the European Medicines Agency has accepted Salix as a herbal medicine product with antiphlogistic effects. It is known, that willow bark can act against chronic pain and inflammation, such as fever with cold, headaches, joint and low back pain. In comparison to Aspirin TM (acetylsalicylic acid; 500 mg -1 g), which inhibits the enzyme COX-2 (cyclooxygenase-2) and the inflammatory prostaglandin production, Salix exhibits fewer side effects and a lower dosage of the extract is needed. This pharmacological efficacy of Salix is thought to occur due to the interaction of many bioactive compounds, such as salicylates, tannins and flavonoids. It has been reported, that salicylates are being hydrolysed into salicin, which is being metabolised in the gut followed by the liver into salicylic acid, making it bioavailable in the human body and showing a pharmacological effect. In the current SaliMed project, the chemical composition of the Salicis cortex extract (willow bark) of different Salix genotypes was investigated and the overall medicinal effect is being analysed.Variable Salix species and crosses have a different chemical composition and grouping them into clusters can help differentiating them. To identify bio-active compounds in willow bark, an inflammatory prostaglandin PGE2-release level analysis in stimulated human immune cells (PBMC) using an ELISA assay can provide useful information about the anti-inflammatory potency of each species, extract, fraction and single compound. The lower the PGE2 release from cells, the better Salix bark extracts could inhibit inflammation.Activity-guided fractionation can offer insides into the most potent fractions and Salix biomarkers can be revealed. Targeted and non-targeted metabolomics can help in identifying known and yet unknown compounds, which can be analysed upon their potency. Therefore, potent Salix bark extract was fractionated by solid-phase extraction (SPE), followed sub-fractionation of the most bio-active SPE fractions using preparative and semi-preparative HPLC. Further, bio-active compounds contained in the fractions were isolated and their structure is going to be determined by NMR.