Inter- and intraspecific diversity of Salix bark phenolic profiles - A resource for the pharmaceutical industry.

Due to their content of phenolic compounds, willow bark preparations are used as an herbal remedy. The large diversity of phenolic secondary metabolites in Salix still provides a resource for the identification of bioactive compounds in particular species, including species not yet in focus from a phytopharmaceutical perspective. The present study describes the bark phenolic profile of 13 Salix species analyzed by HPLC-MS: Salix alba, Salix babylonica, Salix daphnoides, Salix fragilis, Salix hastata, Salix myrsinifolia, Salix pentandra, Salix purpurea, Salix repens (including subspecies S. repens ssp. arenaria and S. repens ssp. repens), Salix rosmarinifolia, Salix sachalinensis, Salix triandra and Salix viminalis. The analyzed profiles comprised the chemical groups of salicylates, flavonoids, procyanidins, phenolic acid derivatives, and some unclassified phenolics. Particular compounds were detected in species where they have not been previously reported. Apart from interspecific diversity, qualitative variability within species was observed as certain components were detected only in some of the analyzed genotypes. The knowledge on specific phenolic profiles of species and genotypes is the basis for the selection of suitable willow bark material with certain desired bioactive properties. Furthermore, the high inter- and intraspecific variability points out the necessity for product standardization of willow bark raw material.

Identification of Salicylates in Willow Bark (Salix Cortex) for Targeting Peripheral Inflammation.

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.

Chemoprofiling as Breeding Tool for Pharmaceutical Use of Salix.

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.

Compound-specific responses of phenolic metabolites in the bark of drought-stressed Salix daphnoides and Salix purpurea.

The bark of willows (Salix spp.) is rich in bioactive phenolic compounds from different compound classes and is therefore used as an herbal remedy. The accumulation of these secondary plant metabolites is influenced by environmental factors, including the availability of water. To analyze the influence of drought stress on the profile of phenolic metabolites in willow bark, a pot experiment with Salix daphnoides Vill. and Salix purpurea L. was conducted. Plants were subjected to three irrigation treatments for four and ten weeks: 65-75% field capacity (well-watered), 33-38% field capacity (moderate drought), and 17-22% field capacity (severe drought). Shoot biomass and proline content were assessed as drought-sensitive traits. Contents of phenolic compounds were analyzed by high-performance liquid chromatography. Drought stress reduced shoot biomass and led to an increase of the bark proline content. The particular effects on phenolics depended on the individual compound, Salix species and drought stress duration. Whereas salicylates were not affected, some flavonoids and phenolic acid derivatives, as well as salireposide indicated treatment effects. The effects comprised decreasing as well as increasing contents. However, beyond the impact of drought stress, the observed responses are assumed to be superimposed by seasonal changes in the content of phenolics. Regarding the yield of willow shoots, the impairment of growth under water shortage seems to be more decisive than drought-induced changes of the bark metabolite content.

Melampsora rust species on biomass willows in central and north-eastern Germany.

Melampsora willow rusts are the most important fungal pathogens in short rotation coppices of biomass willows. In the past, breeding programmes for rust resistant biomass willows concentrated on the distinction of races within the forma specialis Melampsora larici-epitea f. sp. larici-epitea typica that colonized Salix viminalis and related clones. In a new breeding program that is based on a wider range of willow species it is necessary to identify further Melampsora species and formae specialis that are pathogens of willow species other than S. viminalis. Therefore, three stock collections with Salix daphnoides, Salix purpurea, and other shrub willow species (including S. viminalis) species were sampled in north-eastern Germany. A fourth stock collection in central Germany contributed rusts of tree willows (Salix fragilis and Salix alba) and the large shrub Salix caprea. Out of 156 rust samples, 149 were successfully sequenced for ITS rDNA. A phylogenetic analysis combining Neighbour-Joining, Maximum-Likelihood and Bayesian analysis revealed six species: Melampsora ribesii-purpureae, Melampsora allii-salicis-albae, Melampsora sp. aff. allii-fragilis, Melampsora larici-pentandrae, Melampsora larici-caprearum, and Melampsora larici-epitea. The first four species were found exclusively on the expected hosts. Melampsora larici-caprearum had a wider host range comprising S. caprea and S. viminalis hybrids. Melampsora larici-epitea can be further differentiated into two formae speciales. The forma specialis larici-epitea typica (59 samples) colonized Salix viminalis clones, Salix purpurea, Salix×dasyclados, and Salix×aquatica. In contrast to this relatively broad host range, f. sp. larici-daphnoides (65 samples) was found exclusively on Salix daphnoides. With the distinction and identification of the rust species/formae speciales it is now possible to test for race-specific resistances in a more targeted manner within the determined pairings of rust and willow species.

Factors influencing the variability of antioxidative phenolic glycosides in Salix species.

Phenolic glycosides, especially the salicylates, are important secondary metabolites in the bark of willows (Salix spp.). Because of their anti-inflammatory, analgesic, and fever-reducing properties, they are of particular interest to society. Compared to the fabrication of synthetic salicylacetylic acid, the commercial production of willow bark extracts with adequate amounts of salicylate is very difficult due to several biological and technical reasons. Therefore, one of the objectives was to identify salicylate-rich clones from three species, Salix daphnoides , Salix purpurea , and Salix pentandra , with potentially high amounts of phenolic glycosides. Three hundred different Salix clones were collected, and the chemical profiles of their bark were analyzed by HPLC. Overall, S. daphnoides clones showed the highest phenolic glycoside contents, followed by S. purpurea and S. pentandra. Second, seasonal changes of secondary compounds in willow bark were analyzed to determine the optimal harvesting time. The phenolic glycoside levels decreased over the growing season, with highest contents detected during plant dormancy. The effects of different cultivation conditions were also examined, and none of these treatments were found to have a significant effect on the phenolic glycoside content in willow bark. Biomass accumulation in the clones with grass competition was significantly lower than in the other three treatments.

First experiences with contrast-enhanced first-pass MR perfusion imaging in patients with primary, benign cardiac masses and tumour-like lesions.

The aim of this study was to evaluate the diagnostic value of contrast-enhanced first-pass perfusion MRI in patients with suspected cardiac masses and tumour-like lesions. Twenty patients underwent contrast-enhanced first-pass saturation-recovery steady-state-free-precession perfusion MRI in addition to clinical MRI. Eleven diagnostic parameters were analysed blinded in consensus by three observers: localisation (paracardiac/mural/intracavitary), malignancy (benign/malignant) and first-pass enhancement pattern (homogeneous/heterogeneous as well as non-perfused/hypoperfused/iso-perfused/ hyperperfused). The results were compared to combined references comprising histology, cytology, medical and surgical reports, echocardiography, chest X-ray, coronary angiography and regular MRI. Also, we analysed if additional first-pass perfusion confirmed, changed or reduced the number of differential diagnoses compared to clinical MRI. All cardiac masses or tumour-like lesions were correctly localised and scored as benign lesions. For homogeneous perfused lesions the sensitivity, specificity, positive and negative predictive value was 94/100/100/67%, 100/94/67/100% for heterogeneous perfused lesions, 92/100/100/88% for non-perfused, 100/94/75/100 for hypoperfused, 100/100/100/100% for hyperperfused and for isoperfused lesions. In 17/2/1 cases perfusion MRI confirmed, reduced or increased the number of potential differentials. First-pass perfusion MRI provides valuable information in patients with benign cardiac masses or tumour-like lesions. Further experience is needed to underline these preliminary observations.