Background: Reliable human in vitro blood–brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. Methods: Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (C_CL) in real-time. Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. Results: The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. Conclusions: Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products.

Ethnoveterinary knowledge in Europe may play an important role as a basis for sustainable treatment options for livestock. Aims of our study were (a) to compare the ethnoveterinary practices of two culturally and sociodemographically different regions of Switzerland, (b) to compare results with earlier ethnoveterinary studies conducted in Switzerland and in adjacent Italian regions and, (c) to evaluate possible reasons for regional differences in European ethnoveterinary medicine. 25 interviews were conducted in 2014 in all Italian speaking regions (ItR) of Switzerland, and 31 interviews were held in five north-western German speaking Cantons (GeC). Semi-structured questionnaires were used to collect detailed information regarding plant species, mode of preparation, dosage, route of administration, category of use, origin of knowledge, frequency of use, and satisfaction with outcomes of the treatments. A total of 162 homemade remedies in ItR and 219 in GeC were reported, out of which 125 and 145, respectively, were reported to contain only one plant species (homemade single species herbal remedy report, HSHR). 44 ItR and 43 GeC plant species were reported to treat livestock, of which only a half were used in both regions. For each HSHR, we classified the treatment intention of all use reports (UR), leading to a total of 205 and 219 UR in ItR and GeC respectively. While cattle were the most often treated livestock species in both study regions, in ItR 40% of UR were administered to small ruminants. Main indications in both regions were gastrointestinal diseases and skin afflictions, but in ItR a high number of URs were reported as antiparasitics. URs were mainly handed down from the past generation, but in GeC the source of knowledge for 20% of URs were from courses. Regarding the used plant species, ItR showed a higher concordance with Swiss than Italian studies, but with some differences to all regions. A total of 22 (14 ItR; 8 GeC) plant species in this study have not been reported before in ethnoveterinary studies of Swiss and Italian alpine regions. ItR and GeC, show differences and similarities with respect to their own ethnoveterinary practices and earlier Swiss and Italian ethnoveterinary studies. Linguistic, geographical, as well as social and farm-structural conditions influence the regional ethnoveterinary knowledge. However, political borders seem to be more important than language or geographical barriers.

Covering: up to 2012 Since the advent of high-throughput screening (HTS) in the early 1990s, a wealth of innovative technologies have been proposed and implemented for the effective localization and characterization of bioactive constituents in complex matrices. The latest developments in this field are reviewed under the perspective of their applicability to natural product-based drug discovery. The approaches discussed here include TLC-based bioautography, HPLC-based assays with on-line, at-line and off-line detection, as well as affinity-based methods, such as frontal affinity chromatography, pulsed ultrafiltration mass spectrometry, imprinted polymers, and affinity capillary electrophoresis. Selected practical examples are given to illustrate the strengths and limitations of these approaches in contemporary natural product lead discovery. In addition, compatibility issues of natural product extracts and HTS are addressed, and selected protocols for the generation of high quality libraries are presented.

Wound healing is a complex physiological process that is controlled by a well-orchestrated cascade of interdependent biochemical and cellular events, which has spurred the development of therapeutics that simultaneously target these active cellular constituents. We assessed the potential of Parrotia persica (Hamamelidaceae) in wound repair by analyzing the regenerative effects of its two main phenolic compounds, myricetin-3-O-β-rhamnoside and chlorogenic acid. To accomplish this, we performed phytochemical profiling and characterized the chemical structure of pure compounds isolated from P. persica, followed by an analysis of the biological effects of myricetin-3-O-β-rhamnoside and chlorogenic acid on three cell types, including keratinocytes, fibroblasts, and endothelial cells. Myricetin-3-O-β-rhamnoside and chlorogenic acid exhibited complementary pro-healing properties. The percentage of keratinocyte wound closure as measured by a scratch assay was four fold faster in the presence of 10 µg/mL chlorogenic acid, as compared to the negative control. On the other hand, myricetin-3-O-β-rhamnoside at 10 µg/mL was more effective in promoting fibroblast migration, demonstrating a two-fold higher rate of closure compared to the negative control group. Both compounds enhanced the capillary-like tube formation of endothelial cells in an in vitro angiogenesis assay. Our results altogether delineate the potential to synergistically accelerate the fibroblastic and remodelling phases of wound repair by administering appropriate amounts of myricetin-3-O-β-rhamnoside and chlorogenic acid.

Kaempferol is a major flavonoid in the human diet and in medicinal plants. The compound exerts anxiolytic activity when administered orally in mice, while no behavioural changes were observed upon intraperitoneal administration, or upon oral administration in gut sterilized animals. 4-Hydroxyphenylacetic acid (4-HPAA), which possesses anxiolytic effects when administered intraperitoneally, is a major intestinal metabolite of kaempferol. Pharmacokinetic properties of the compounds are currently not clear. UHPLC-MS/MS methods were validated to support pharmacokinetic studies of kaempferol and 4-HPAA in rats. Non-compartmental and compartmental analyses were performed. After intravenous administration, kaempferol followed a one-compartment model, with a rapid clearance (4.40-6.44l/h/kg) and an extremely short half-life of 2.93-3.79min. After oral gavage it was not possible to obtain full plasma concentration-time profiles of kaempferol. Pharmacokinetics of 4-HPAA was characterized by a two-compartment model, consisting of a quick distribution phase (half-life 3.04-6.20min) followed by a fast elimination phase (half-life 19.3-21.1min). Plasma exposure of kaempferol is limited by poor oral bioavailability and extensive metabolism. Both compounds are rapidly eliminated, so that effective concentrations at the site of action do not appear to be reached. At present, it is not clear how the anxiolytic-like effects reported for the compounds can be explained.