Phytochemical analysis and anthelmintic activity of Combretum mucronatum leaf extract against infective larvae of soil-transmitted helminths including ruminant gastrointestinal nematodes.

BACKGROUND: Soil-transmitted helminths (STH) infect more than a quarter of the world's human population. In the absence of vaccines for most animal and human gastrointestinal nematodes (GIN), treatment of infections primarily relies on anthelmintic drugs, while resistance is a growing threat. Therefore, there is a need to find alternatives to current anthelmintic drugs, especially those with novel modes of action. The present work aimed to study the composition and anthelmintic activity of Combretum mucronatum leaf extract (CMLE) by phytochemical analysis and larval migration inhibition assays, respectively. METHODS: Combretum mucronatum leaves were defatted with petroleum ether and the residue was extracted by ethanol/water (1/1) followed by freeze-drying. The proanthocyanidins and flavonoids were characterized by thin layer chromatography (TLC) and ultra-high performance liquid chromatography (UPLC). To evaluate the inhibitory activity of this extract, larval migration assays with STH and GIN were performed. For this purpose, infective larvae of the helminths were, if necessary, exsheathed (Ancylostoma caninum, GIN) and incubated with different concentrations of CMLE. RESULTS: CMLE was found to be rich in flavonoids and proanthocyanidins; catechin and epicatechin were therefore quantified for standardization of the extract. Data indicate that CMLE had a significant effect on larval migration. The effect was dose-dependent and higher concentrations (1000 µg/mL) exerted significantly higher larvicidal effect (P < 0.001) compared with the negative control (1% dimethyl sulfoxide, DMSO) and lower concentrations (≤ 100 µg/ml). Infective larvae of Ascaris suum [half-maximal inhibitory concentration (IC50) = 5.5 µg/mL], Trichuris suis (IC50 = 7.4 µg/mL), and A. caninum (IC50 = 18.9 µg/mL) were more sensitive to CMLE than that of Toxocara canis (IC50 = 310.0 µg/mL), while infective larvae of Toxocara cati were largely unaffected (IC50 > 1000 µg/mL). Likewise, CMLE was active against most infective larvae of soil-transmitted ruminant GIN, except for Cooperia punctata. Trichostrongylus colubriformis was most sensitive to CMLE (IC50 = 2.1 µg/mL) followed by Cooperia oncophora (IC50 = 27.6 µg/mL), Ostertagia ostertagi (IC50 = 48.5 µg/mL), Trichostrongylus axei (IC50 = 54.7 µg/mL), Haemonchus contortus (IC50 = 145.6 µg/mL), and Cooperia curticei (IC50 = 156.6 µg/mL). CONCLUSIONS: These results indicate that CMLE exhibits promising anthelmintic properties against infective larvae of a large variety of soil-transmitted nematodes.

Rationalising Optimal Dosing of Phytotherapeutics For Use In Children: Current Status - Potential Solutions - Actions Needed.

"Children are not small adults with respect to the treatment with medicinal products." This statement of the WHO was the basis for the initiative of the European Commission for the establishment of a paediatric regulation in 2007 to improve the health of children by facilitating the development of medicines for children and adolescents. Seventeen years later, in the field of herbal medicinal products, results are still sobering. Therefore, the Foundation Plants for Health, Society for Medicinal Plants and Natural Products Research, and German Society for Phytotherapy organised a symposium to assess the status quo for the paediatric use of herbal medicinal products (HMPs), to analyse the causes of the current situation, and to discuss strategies for establishing the proof of safe and efficacious HMPs for children.The current situation for HMPs and their use in children is not fulfilling the requirements of legislation. HMPs in paediatrics are effective and safe, but considering the needs of children is necessary. In European countries, the use, registration, and marketing of HMPs are different, depending on the respective national regulations and specific traditions. EU herbal monographs are the best common denominator for such procedures. Emerging safety discussions must be considered. New approaches with real-world data might be a solution. The regulatory framework is to be adapted. Defining rationalised dosing for HMPs can be achieved by the extrapolation of data from adults, by using existing clinical data for children, and by using RWD. Therefore, a strong need for revising restrictions for the use of HMPs in children and rationalising defined dosage regimes is obvious.

High molecular/low acetylated chitosans reduce adhesion of Campylobacter jejuni to host cells by blocking JlpA.

Infections caused by Campylobacter spp. are a major cause of severe enteritis worldwide. Multifactorial prevention strategies are necessary to reduce the prevalence of Campylobacter. In particular, antiadhesive strategies with specific inhibitors of early host-pathogen interaction are promising approaches to reduce the bacterial load. An in vitro flow cytometric adhesion assay was established to study the influence of carbohydrates on the adhesion of C. jejuni to Caco-2 cells. Chitosans with a high degree of polymerization and low degree of acetylation were identified as potent antiadhesive compounds, exerting significant reduction of C. jejuni adhesion to Caco-2 cells at non-toxic concentrations. Antiadhesive and also anti-invasive effects were verified by confocal laser scanning microscopy. For target identification, C. jejuni adhesins FlpA and JlpA were expressed in Escherichia coli ArcticExpress, and the influence of chitosan on binding to fibronectin and HSP90α, respectively, was investigated. While no effects on FlpA binding were found, a strong inhibition of JlpA-HSP90α binding was observed. To simulate real-life conditions, chicken meat was inoculated with C. jejuni, treated with antiadhesive chitosan, and the bacterial load was quantified. A strong reduction of C. jejuni load was observed. Atomic force microscopy revealed morphological changes of C. jejuni after 2 h of chitosan treatment, indicating disturbance of the cell wall and sacculi formation by electrostatic interaction of positively charged chitosan with the negatively charged cell surface. In conclusion, our data indicate promising antiadhesive and anti-invasive potential of high molecular weight, strongly de-acetylated chitosans for reducing C. jejuni load in livestock and food production. KEY POINTS: • Antiadhesive effects of chitosan with high DP/low DA against C. jejuni to host cells • Specific targeting of JlpA/Hsp90α interaction by chitosan • Meat treatment with chitosan reduces C. jejuni load.

Host-pathogen interaction: Enterobacter cloacae exerts different adhesion and invasion capacities against different host cell types.

New antibiotics are urgently needed due to the huge increase of multidrug-resistant bacteria. The underexplored gram-negative bacterium Enterobacter cloacae is known to cause severe urinary tract and lung infections (UTIs). The pathogenicity of E. cloacae in UTI has only been studied at the bioinformatic level, but until now not within systematic in vitro investigations. The present study assesses different human cell lines for monitoring the early steps of host-pathogen interaction regarding bacterial adhesion to and invasion into different host cells by flow cytometric adhesion assay, classical cell counting assay, gentamicin invasion assay, and confocal laser scanning microscopy. To our knowledge, this is the first report in which E. cloacae has been investigated for its interaction with human bladder, kidney, skin, and lung cell lines under in vitro conditions. Data indicate that E. cloacae exerts strong adhesion to urinary tract (bladder and kidney) and lung cells, a finding which correlates with the clinical relevance of the bacterium for induction of urinary tract and lung infections. Furthermore, E. cloacae ATCC 13047 barely adheres to skin cells (A-431) and shows no relevant interaction with intestinal cells (Caco-2, HT-29), even in the presence of mucin (HT29 MTX). In contrast, invasion assays and confocal laser scanning microscopy demonstrate that E. cloacae internalizes in all tested host cells, but to a different extent. Especially, bladder and kidney cells are being invaded to the highest extent. Defective mutants of fimH and fimA abolished the adhesion of E. cloacae to T24 cells, while csgA deletion had no influence on adhesion. These results indicate that E. cloacae has different pattern for adhesion and invasion depending on the target tissue, which again correlates with the clinical relevance of the pathogen. For detailed investigation of the early host-pathogen interaction T24 bladder cells comprise a suitable assay system for evaluation the bacterial adhesion and invasion.

Tamm-Horsfall protein in humane urine: sex-dependent differences in the excretion and N-glycosylation pattern.

Tamm-Horsfall protein (THP) is a highly N-glycosylated protein from epithelial cells of the ascending limb of Henle loop. It is secreted into the urine as part of the innate immune response against uropathogenic pathogens. As women are more likely to suffer from urinary tract infections, biomedical studies were conducted to investigate sex-differences in THP excretion, as well as differences in the THP N-glycosylation pattern. A total of 238 volunteers (92 men, 146 women, 69 with hormonal contraceptives) participated in this study, providing urine samples. Women showed a clear tendency to have higher THP concentration and excretion rates than men (p < 0.16). Regular intake of hormonal contraceptives had no significant influence on urinary THP concentration compared to no regular intake. The individual N-glycosylation pattern of THP in urine samples from randomly selected individuals (10 female, 10 male) was investigated after enzymatic release and MS analysis of the oligosaccharides. Female subjects tended to have an increased proportion of oligomannose type N-glycans and non-fucosylated glycans, whereas men had an increased proportion of fucosylated complex-type glycans. The higher level of oligomannose-type glycans in THP from women might be explained by a self-defence mechanism to overcome the higher infections pressure by the female anatomical properties.

Towards the development of analytical monograph specifications for the quality assessment of the medicinal plant Phyllanthus urinaria.

Many people in developing countries rely on herbal remedies for their primary healthcare needs. The challenge however is that several of these products lack proper documentation of quality and safety. To ensure consistent quality, validated methods are needed to establish and control quality attributes associated with identity, purity, and levels of bioactive constituents of the respective herbal materials. The present study focused on Phyllanthus urinaria (PU), a widely used medicinal plant in Ghana and West Africa that lacks the necessary quality control standards. The study aimed to develop an HPTLC identification method, which together with UPLC-ESI-Q-TOF-MS/MS analysis established the identity of PU samples and differentiated PU from other closely related Phyllanthus species. Quantitative UPLC and HPTLC methods were developed to assess the contents of selected active markers in the PU samples, which invariably led to the proposal of acceptance criteria for the active markers. Prior to the content analyses, the sample extraction procedure was optimized through the use of Design of Experiment method. The effects of harvest time and geographic origin on the content of active compounds were demonstrated in the investigations. PU samples were also found to be contaminated with higher levels of pesticides like chlorpyrifos and folpet. Essentially, this study provides analytical protocols, insights into the quality status of PU samples in Ghana, and analytical specifications contained in a drafted monograph for future consideration in regional and subregional African pharmacopoeias.

Anthelmintic Activities of Extract and Ellagitannins from Phyllanthus urinaria against Caenorhabditis elegans and Zoonotic or Animal Parasitic Nematodes.

The aerial parts of Phyllanthus urinaria are used in traditional medicine in West Africa against helminthiasis, but their anthelmintic potential has not been evaluated until now. Within the current study, a hydroacetonic extract (AWE) and fractions and isolated ellagitannins from P. urinaria were, therefore, tested in vitro against Caenorhabditis elegans and the larvae of the animal parasites Toxocara canis, Ascaris suum, Ancylostoma caninum, and Trichuris suis. Compounds 1:  - 13: , mainly representing ellagitannins, were isolated using different chromatographic methods, and their structures were elucidated by HR-MS and 1H/13C-NMR. AWE exerted concentration-dependent lethal effects (LC50 of 2.6 mg/mL) against C. elegans and inhibited larval migration of all animal parasites tested (T. suis L1 IC50 24.3 µg/mL, A. suum L3 IC50 35.7 µg/mL, A. caninum L3 IC50 112.8 µg/mL, T. canis L3 IC50 1513.2 µg/mL). The anthelmintic activity of AWE was mainly related to the polar, tannin-containing fractions. Geraniin 1: , the major ellagitannin in the extract, showed the strongest anthelmintic activity in general (IC50 between 0.6 and 804 µM, depending on parasite species) and was the only compound active against A. caninum (IC50 of 35.0 µM). Furosin 9: was least active despite structural similarities to 1: . Among the parasites tested, Trichuris suis L1 larvae turned out to be most sensitive with IC50 of 0.6, 6.4, 4.0, 4.8, and 2.6 µM for geraniin 1: , repandusinic acid A 3: , punicafolin 8: , furosin 9: , and phyllanthusiin A 10: , respectively.

Campylobacter jejuni: targeting host cells, adhesion, invasion, and survival.

Campylobacter jejuni, causing strong enteritis, is an unusual bacterium with numerous peculiarities. Chemotactically controlled motility in viscous milieu allows targeted navigation to intestinal mucus and colonization. By phase variation, quorum sensing, extensive O-and N-glycosylation and use of the flagellum as type-3-secretion system C. jejuni adapts effectively to environmental conditions. C. jejuni utilizes proteases to open cell-cell junctions and subsequently transmigrates paracellularly. Fibronectin at the basolateral side of polarized epithelial cells serves as binding site for adhesins CadF and FlpA, leading to intracellular signaling, which again triggers membrane ruffling and reduced host cell migration by focal adhesion. Cell contacts of C. jejuni results in its secretion of invasion antigens, which induce membrane ruffling by paxillin-independent pathway. In addition to fibronectin-binding proteins, other adhesins with other target structures and lectins and their corresponding sugar structures are involved in host-pathogen interaction. Invasion into the intestinal epithelial cell depends on host cell structures. Fibronectin, clathrin, and dynein influence cytoskeletal restructuring, endocytosis, and vesicular transport, through different mechanisms. C. jejuni can persist over a 72-h period in the cell. Campylobacter-containing vacuoles, avoid fusion with lysosomes and enter the perinuclear space via dynein, inducing signaling pathways. Secretion of cytolethal distending toxin directs the cell into programmed cell death, including the pyroptotic release of proinflammatory substances from the destroyed cell compartments. The immune system reacts with an inflammatory cascade by participation of numerous immune cells. The development of autoantibodies, directed not only against lipooligosaccharides, but also against endogenous gangliosides, triggers autoimmune diseases. Lesions of the epithelium result in loss of electrolytes, water, and blood, leading to diarrhea, which flushes out mucus containing C. jejuni. Together with the response of the immune system, this limits infection time. Based on the structural interactions between host cell and bacterium, the numerous virulence mechanisms, signaling, and effects that characterize the infection process of C. jejuni, a wide variety of targets for attenuation of the pathogen can be characterized. The review summarizes strategies of C. jejuni for host-pathogen interaction and should stimulate innovative research towards improved definition of targets for future drug development. KEY POINTS: • Bacterial adhesion of Campylobacter to host cells and invasion into host cells are strictly coordinated processes, which can serve as targets to prevent infection. • Reaction and signalling of host cell depend on the cell type. • Campylobacter virulence factors can be used as targets for development of antivirulence drug compounds.

Microbiological and Clinical Effects of a Proanthocyanidin-enriched Extract from Rumex acetosa in Periodontally Healthy Carriers of Porphyromonas gingivalis: a Randomized Controlled Pilot Study.

Rumex acetosa significantly inhibits the adhesion of Porphyromonas gingivalis (P. g.) to eukaryotic host cells in vitro. The objective of this randomized placebo-controlled pilot-trial was to analyze effects of a mouth rinse containing 0.8% (w/w) of a quantified proanthocyanidin-enriched extract from Rumex acetosa (RA1) on microbiological, clinical, and cytological parameters in systemically healthy individuals without history of periodontitis, harboring P. g. intraorally. 35 subjects received a supragingival debridement (SD) followed by mouth rinsing (3 times daily) with either RA1 mouth rinse solution (test) or placebo (control) for 7 days as adjunct to routine oral hygiene. Supragingival biofilm samples were taken at screening visit, baseline (BL), 2, 4, 7 and 14 days after SD. P. g. and 11 other oral microorganisms were detected and quantified by rtPCR. Changes in the oral microbiota composition of one test and one control subject were assessed via high throughput 16S rRNS gene amplicon sequencing. Approximal Plaque Index (API) and the modified Sulcular Bleeding Index (SBI) were assessed at BL, 7- and 14-days following SD. Brush biopsies were taken at BL and 14 d following SD. Intergroup comparisons revealed no significant microbiological, cytological, and clinical differences at any timepoint. However, a significant reduction in SBI at day 14 (p = 0.003) and API at day 7 (p = 0.02) and day 14 (p = 0.009) was found in the test group by intragroup comparison. No severe adverse events were observed. The results indicate that RA1 mouth rinse is safe but does not seem to inhibit colonization of P. g. or improve periodontal health following SD.

Seven-day Oral Intake of Orthosiphon stamineus Leaves Infusion Exerts Antiadhesive Ex Vivo Activity Against Uropathogenic E. coli in Urine Samples.

Orthosiphon stamineus leaves (Java tea) extract is traditionally used for the treatment of urinary tract infections. According to recent in vitro data, animal infection studies, and transcriptomic investigations, polymethoxylated flavones from Java tea exert antiadhesive activity against uropathogenic Escherichia coli (UPEC). This antiadhesive activity has been shown to reduce bladder and kidney lesion in a mice infection model. As no data on the antivirulent activity of Java tea intake on humans are available, a biomedical study was performed on 20 healthy volunteers who self-administered Orthosiphon infusion (4 × 3 g per day, orally) for 7 days. The herbal material used for the study conformed to the specification of the European Pharmacopoeia, and ultra high-performance liquid chromatography (UHPLC) of the infusion showed rosmarinic acid, caffeic acid, and cichoric acid to be the main compounds aside from polymethoxylated flavones. Rosmarinic acid was quantified in the tea preparations with 243 ± 22 µg/mL, indicating sufficient reproducibility of the preparation of the infusion. Urine samples were obtained during the biomedical study on day 1 (control urine, prior to Java tea intake), 3, 6 and 8. Antiadhesive activity of the urine samples was quantified by flowcytometric assay using pre-treated UPEC NU14 and human T24 bladder cells. Pooled urine samples indicated significant inhibition of bacterial adhesion on day 3, 6 and 8. The urine samples had no influence on the invasion of UPEC into host cells. Bacterial proliferation was slightly reduced after 24 h incubation with the urine samples. Gene expression analysis (qPCR) revealed strong induction of fitness and motility gene fliC and downregulation of hemin uptake system chuT. These data correlate with previously reported datasets from in vitro transcriptomic analysis. Increased bacterial motility was monitored using a motility assay in soft agar with UPEC UTI89. The intake of Java tea had no effect on the concentration of Tamm-Horsfall Protein in the urine samples. The present study explains the antiadhesive and anti-infective effect of the plant extract by triggering UPEC from a sessile lifestyle into a motile bacterial form, with reduced adhesive capacity.

Microstructured Polymer System Containing Proanthocyanidin-Enriched Extract from Limonium brasiliense as a Prophylaxis Strategy to Prevent Recurrence of Porphyromonas gingivalis.

Periodontal diseases are a global oral health problem affecting almost 10% of the global population. Porphyromonas gingivalis is one of the main bacteria involved in the initiation and progression of inflammatory processes as a result of the action of the cysteine proteases lysin- and arginine-gingipain. Surelease/polycarbophil microparticles containing a lyophilized proanthocyanidin-enriched fraction from the rhizomes of Limonium brasiliense, traditionally named "baicuru" (ethyl acetate fraction), were manufactured. The ethyl acetate fraction was characterized by UHPLC by the presence of samarangenins A and B (12.10 ± 0.07 and 21.05 ± 0.44%, respectively) and epigallocatechin-3-O-gallate (13.44 ± 0.27%). Physiochemical aspects of Surelease/polycarbophil microparticles were characterized concerning particle size, zeta potential, entrapment efficiency, ethyl acetate fraction release, and mucoadhesion. Additionally, the presence of the ethyl acetate fraction-loaded microparticles was performed concerning potential influence on viability of human buccal KB cells, P. gingivalis adhesion to KB cells, gingipain activity, and P. gingivalis biofilm formation. In general, all Surelease/polycarbophil microparticles tested showed strong adhesion to porcine cheek mucosa (93.1 ± 4.2% in a 30-min test), associated with a prolonged release of the ethyl acetate fraction (up to 16.5 ± 0.8% in 24 h). Preincubation of KB cells with Surelease/polycarbophil microparticles (25 µg/mL) resulted in an up to 93 ± 2% reduced infection rate by P. gingivalis. Decreased activity of the P. gingivalis-specific virulence factors lysin- and arginine-gingipain proteases by Surelease/polycarbophil microparticles was confirmed. Surelease/polycarbophil microparticles decreased biofilm formation of P. gingivalis (97 ± 2% at 60 µg/mL). Results from this study prove the promising activity of Surelease/polycarbophil microparticles containing ethyl acetate fraction microparticles as a prophylaxis strategy to prevent the recurrence of P. gingivalis.

Development of an Analytical Workflow to Support the Establishment of Monographs in African Pharmacopoeias - Combretum mucronatum Leaves as Example.

Herbal medicines are invaluable in African medicine, but quality and safety are not documented in many cases. Besides controlled farming, validated quality control methods are needed to ensure identity, purity, and content. Analytical specifications within modern monographs are needed for consistent batch quality. Combretum mucronatum leaves are widely used in West Africa, but state-of-the-art quality control methods and specifications are non-existent. The aim of the following study was the development of ICH-validated chromatographic protocols for identity, purity, content assay, and analytical specifications for consideration into pharmacopoeial monographs. UPLC-ESI-Q-TOF-MS/MS was used for untargeted phytochemical information on composition. Optimisation of extraction was based on phytochemical profiling. HPTLC was used for differentiation of C. mucronatum from other Combretum species and UPLC for simultaneous determination of 7 marker compounds. C. mucronatum batch analyses (n = 49) investigated the influence of harvest time and geographical origin. Pesticides screening from a 349-compound panel were carried out. 30 compounds, identified by LC-MS, were used for characterization of the plant material. Orietin, isoorientin, vitexin and isovitexin were used as specific marker compounds for qualitative and quantitative HPTLC purposes, while UPLC quantified additionally epicatechin, procyanidins B2 and C1. Influence of harvest time and geographic origin on the content of marker compounds was observed. Differences in the metabolite profiles of C. mucronatum compared to related Combretum species were established for quality control purposes. Contamination with high amoounts of chlorpyrifos, and folpet (sum of folpet and phtalimide, expressed as folpet) were also observed.The study provides analytical protocols, analytical specifications and a drafted monograph for consideration for African pharmacopoeias, and reveals potential challenges in the quality of C. mucronatum.

Effects of Extracts and Flavonoids from Drosera rotundifolia L. on Ciliary Beat Frequency and Murine Airway Smooth Muscle.

Extracts from Drosera rotundifolia are traditionally used to treat cough symptoms during a common cold. The present study aimed to investigate the impact of extracts from D. rotundifolia and active compounds on the respiratory tract. Tracheal slices of C57BL/6N mice were used ex vivo to examine effects on airway smooth muscle (ASM) and ciliary beat frequency (CBF). Phosphodiesterase (PDE) inhibition assays were carried out to test whether PDE1 or PDE4 are targeted by the active compounds. An ethanol-water extract, as well as an aqueous fraction of this extract, exerted antispasmodic properties against acetylcholine-induced contractions. In addition, contractions induced by 60 mM K+ were abrogated by the aqueous fraction. Effects on ASM could be attributed to the flavonoids quercetin, 2″-O-galloylhyperoside and hyperoside. Moreover, the Drosera extract and the aqueous fraction increased the CBF of murine tracheal slices. Quercetin and 2″-O-galloylhyperoside were identified as active compounds involved in the elevation of CBF. Both compounds inhibited PDE1A and PDE4D. The elevation of CBF was mimicked by the subtype-selective PDE inhibitor rolipram (PDE4) and by 8-methoxymethyl-IBMX. In summary, our study shows, for the first time, that a Drosera extract and its flavonoid compounds increase the CBF of murine airways while antispasmodic effects were transferred to ASM.

Phytochemical and functional analysis of horseradish (Armoracia rusticana) fermented and non-fermented root extracts.

BACKGROUND: The roots of horseradish (Armoracia rusticana) are used for infections of respiratory airway and for urinary tract infections due to isothiocyanates (ITC), enzymatically formed during fermentation of glucosinolates by myrosinase. HYPOTHESIS/PURPOSE: The present study aims to present a comprehensive overview on the phytochemical composition of A. rusticana roots, especially concerning isothiocyanates and respective glucosinolates. The complex flavonoid spectrum of the herbal material is reviewed. Published data on in vitro activity of horseradish extracts and isolated compounds are summarized. These data indicate well-established use of horseradish as an antibacterial remedy against bacterial infections of the airway and urinary tract. STUDY DESIGN: To answer the question if other compounds from A. rusticana beside ITC contribute to the antibacterial activity, non-targeted LC-MS studies were performed with fermented and non-fermented horseradish extracts, and detailed phytochemical profiles were established. RESULTS: Comparative investigations on the antibacterial activity indicated that only ITC-containing extracts and fractions exert antibacterial activity. The huge variety of non-ITC compounds do not significantly contribute to the antibacterial activity, but can be used for analytical characterisation and quality control of the herbal material. Detailed phytochemical analysis additionally revealed a variety of compounds, not described until now for horseradish roots: the flavonol glycosides kaempferol-3-O-ß-d-xylopyranosyl-(1''' â†’ 2'')-ß-d-galactopyranoside, kaempferol-3-O-α-l-rhamnopyranosyl-(1''' â†’ 6'')-ß-d-glucopyranoside, kaempferol-3-O-ß-d-glucopyranoside, Kaempferol-3-O-ß-d-xylopyranosyl-7-O-ß-d-glucopyranoside, Kaempferol-3-O-ß-d-xylopyranosyl-(1'''' â†’ 2''')-ß-d-galactopyranoside-7-O-ß-d-glucopyranoside, the oxo-indole derivative spirobrassinin, the phenylthiazole 2-methylsulfanyl-4-phenyl-4,5-dihydro-1,3-thiazole, a series of lysophophatidylethanolamine and 13 different N-phenylpropenoyl-L-amino acids. CONCLUSION: The antibacterial effects of horseradish are only due to the presence of glucosinolates resp. the corresponding ITC, and the detailed overall composition of horseradish extracts has been reported.

Quality assessment of African herbal medicine: A systematic review and the way forward.

BACKGROUND: In Africa, herbalism supplements allopathic medicine's efforts to ensure Universal Health Coverage attainment. This review was conducted to identify and to summarise current literature on methodological approaches used for quality control of herbal medicines in Africa, to evaluate the gaps associated with existing strategies within context of best practices, and make recommendations for future improvements. METHODS: A systematic search was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Articles were screened and assessed for eligibility. RESULTS: 118 articles were included into the study. There was a high preference for impurity profiling tests (77%) indicating the prioritization for tests that guarantee safety despite the limited analytical resources available. Other classes of tests reported included identification tests (29%), physicochemical tests (18%), and content assays (12%). Although standard methods exist in preparing samples for impurity tests, different techniques were observed in different studies, and this could lead to differences in analytical outcomes. Content assays focused on single marker assessments, which may be inadequate to comprehensively assess the quality of products. CONCLUSION: This review provides knowledge of existing strengths and challenges for herbal medicine quality assessments in Africa. For future it is recommended to implement more studies on contaminants (e.g. mycotoxins) and pharmaceutical adulterants. The use of chemometrics to develop analytical methods should be promoted. Also, stakeholders in the medicine quality industry in Africa need to effectively collaborate to establish a well co-ordinated and harmonized system to provide a sustainable framework for the GACP and GMP guided production and quality assurance of herbal medicines.

Aqueous extract from Equisetum arvense stimulates the secretion of Tamm-Horsfall protein in human urine after oral intake.

BACKGROUND: Within European traditional phytotherapy, extracts from different herbal plants are used for prevention and therapy of uncomplicated urinary tract infections and for flushing out of kidney grits. Besides increased urine flow by slight diuretic effects, also stimulation of Tamm-Horsfall protein (syn. THP, uromodulin) in the distal part of the kidney could explain reduced kidney gravel and anti-virulent activity against uropathogenic E. coli. PURPOSES: Evaluation of THP-inducing activity of extracts from Equisetum arvense, Levisticum officinalis, Ilex paraguariensis, Juniperus communis, Urtica dioica, and Taraxacum officinale by quantification of THP in urine samples after oral application to humans. STUDY DESIGN: 7 days p.o. application of the test intervention to healthy volunteers (n = 10 per intervention group) and analysis of urine samples at day 1 (untreated control values), and days 3, 6 and 8 on THP content by validated ELISA. Antiadhesive activity of urine samples was monitored by flow cytometry using UPEC strain NU14 against human T24 bladder cells. RESULTS: An aqueous extract from E. arvense, fully characterized by a specific LC-MS method, induced THP concentration in urine samples significantly during a 7-day p.o. application up to 300%, related to the untreated controls. Ex vivo investigation of the individual and pooled urine samples with elevated THP concentrations showed good correlation to antiadhesive effects against UPEC NU14 to T24 cells. Urine samples of the Equisetum treated volunteers had no effect on the proliferation and on biofilm formation of UPEC NU14. Silica excretion in the urine samples had no correlation to the respective THP levels. Monitoring of electrolyte content in the urine samples indicat ed diuretic effects of the intervention with Equisetum extract. Detailed phytochemical analysis of the Equisetum extract by LC-MS and LC-UV revealed an analytical protocol, which identified > 80 compounds from the extract by MS evaluations and 18 compounds by UV detection. This protocol will provide a valuable tool for future quality control of Equisetum extract. CONCLUSION: Aqueous extract from E. arvense significantly stimulates THP secretion in urine samples after 7 days of oral intake and inhibits the interplay between UPEC and bladder host cells. This could explain the therapeutic use of this herbal material for urinary tract infections and kidney gravel. Detailed phytochemical analysis of the Equisetum extract by LC-MS and LC-UV revealed an analytical protocol, which identified > 82% of all eluted compounds. This protocol will provide a valuable tool for future quality control of Equisetum extract.

Broadly Applicable, Virus-Free Dual Reporter Assay to Identify Compounds Interfering with Membrane Fusion: Performance for HSV-1 and SARS-CoV-2.

Membrane fusion constitutes an essential step in the replication cycle of numerous viral pathogens, hence it represents an important druggable target. In the present study, we established a virus-free, stable reporter fusion inhibition assay (SRFIA) specifically designed to identify compounds interfering with virus-induced membrane fusion. The dual reporter assay is based on two stable Vero cell lines harboring the third-generation tetracycline (Tet3G) transactivator and a bicistronic reporter gene cassette under the control of the tetracycline responsive element (TRE3G), respectively. Cell-cell fusion by the transient transfection of viral fusogens in the presence of doxycycline results in the expression of the reporter enzyme secreted alkaline phosphatase (SEAP) and the fluorescent nuclear localization marker EYFPNuc. A constitutively expressed, secreted form of nanoluciferase (secNLuc) functioned as the internal control. The performance of the SRFIA was tested for the quantification of SARS-CoV-2- and HSV-1-induced cell-cell fusion, respectively, showing high sensitivity and specificity, as well as the reliable identification of known fusion inhibitors. Parallel quantification of secNLuc enabled the detection of cytotoxic compounds or insufficient transfection efficacy. In conclusion, the SRFIA reported here is well suited for high-throughput screening for new antiviral agents and essentially will be applicable to all viral fusogens causing cell-cell fusion in Vero cells.