Willow Bark-Derived Material with Antibacterial and Antibiofilm Properties for Potential Wound Dressing Applications.

Tree stems contain wood in addition to 10-20% bark, which remains one of the largest underutilized biomasses on earth. Unique macromolecules (like lignin, suberin, pectin, and tannin), extractives, and sclerenchyma fibers form the main part of the bark. Here, we perform detailed investigation of antibacterial and antibiofilm properties of bark-derived fiber bundles and discuss their potential application as wound dressing for treatment of infected chronic wounds. We show that the yarns containing at least 50% of willow bark fiber bundles significantly inhibit biofilm formation by wound-isolated Staphylococcus aureus strains. We then correlate antibacterial effects of the material to its chemical composition. Lignin plays the major role in antibacterial activity against planktonic bacteria [i.e., minimum inhibitory concentration (MIC) 1.25 mg/mL]. Acetone extract (unsaturated fatty acid-enriched) and tannin-like (dicarboxylic acid-enriched) substances inhibit both bacterial planktonic growth [MIC 1 and 3 mg/mL, respectively] and biofilm formation. The yarn lost its antibacterial activity once its surface lignin reached 20.1%, based on X-ray photoelectron spectroscopy. The proportion of fiber bundles at the fabricated yarn correlates positively with its surface lignin. Overall, this study paves the way to the use of bark-derived fiber bundles as a natural-based material for active (antibacterial and antibiofilm) wound dressings, upgrading this underappreciated bark residue from an energy source into high-value pharmaceutical use.

Effect of Hybrid Type and Harvesting Season on Phytochemistry and Antibacterial Activity of Extracted Metabolites from Salix Bark.

Hundreds of different fast-growing Salix hybrids have been developed mainly for energy crops. In this paper, we studied water extracts from the bark of 15 willow hybrids and species as potential antimicrobial additives. Treatment of ground bark in water under mild conditions extracted 12-25% of the dry material. Preparative high-performance liquid chromatography is proven here as a fast and highly efficient tool in the small-scale recovery of raffinose from Salix bark crude extracts for structural elucidation. Less than half of the dissolved material was assigned by chromatographic (gas chromatography and liquid chromatography) and spectroscopic (mass spectrometry and nuclear magnetic resonance spectroscopy) techniques for low-molecular-weight compounds, including mono- and oligosaccharides (sucrose, raffinose, and stachyose) and aromatic phytochemicals (triandrin, catechin, salicin, and picein). The composition of the extracts varied greatly depending on the hybrid or species and the harvesting season. This information generated new scientific knowledge on the variation in the content and composition of the extracts between Salix hybrids and harvesting season depending on the desired molecule. The extracts showed high antibacterial activity on Staphylococcus aureus with a minimal inhibitory concentration (MIC) of 0.6-0.8 mg/mL; however, no inhibition was observed against Escherichia coli, Enterococcus faecalis, and Salmonella typhimurium. MIC of triandrin (i.e., 1.25 mg/mL) is reported for the first time. Although antibacterial triandrin and (+)-catechin were present in extracts, clear correlation between the antibacterial effect and the chemical composition was not established, which indicates that antibacterial activity of the extracts mainly originates from some not yet elucidated substances. Aquatic toxicity and mutagenicity assessments showed the safe usage of Salix water extracts as possible antibacterial additives.

Development and validation of a high-content screening assay for inhibitors of enteropathogenic E. coli adhesion.

Enteropathogenic E. coli (EPEC) causes intestinal infections leading to severe diarrhea. EPEC attaches to the host cell causing lesions to the intestinal epithelium coupled with the effacement of microvilli. In the process, actin accumulates into a pedestal-like structure under bacterial microcolonies. We designed an automated fluorescence microscopy-based screening method for discovering compounds capable of inhibiting EPEC adhesion and virulence using aurodox, a type three secretion system (T3SS) inhibitor, as a positive control. The screening assay employs an EPEC strain (2348/69) expressing a fluorescent protein and actin staining for monitoring the bacteria and their pedestals respectively, analyzing these with a custom image analysis pipeline. The assay allows for the discovery of compounds capable of preventing the formation of pathogenic actin rearrangements. These compounds may be interfering with virulence-related molecular pathways relevant for developing antivirulence leads.

Composition and Antibacterial Effect of Mint Flavorings in Candies and Food Supplements.

Mint flavorings are widely used in confections, beverages, and dairy products. For the first time, mint flavoring composition of mint candies and food supplements (n = 45), originating from 16 countries, as well as their antibacterial properties, was analyzed. The flavorings were isolated by Marcusson's type micro-apparatus and analyzed by GC-MS. The total content of the mint flavoring hydrodistilled extracts was in the range of 0.01 - 0.9%. The most abundant compounds identified in the extracts were limonene, 1,8-cineole, menthone, menthofuran, isomenthone, menthol and its isomers, menthyl acetate. The antimicrobial activity of 13 reference substances and 10 selected mint flavoring hydrodistilled extracts was tested on Escherichia coli and Staphylococcus aureus by broth dilution method. Linalool acetate and (-)-carvone, as most active against both bacteria, had the lowest MIC90 values. (+)-Menthyl acetate, (-)-menthyl acetate, and limonene showed no antimicrobial activity. Three of the tested extracts had antimicrobial activity against E. coli and 8 extracts against S. aureus. Their summary antimicrobial activity was not always in concordance with the activities of respective reference substances.

Structure-Based Virtual Screening of LsrK Kinase Inhibitors to Target Quorum Sensing.

In the era of increased antibiotic resistance, targeting enzymes involved in bacterial communication (quorum sensing) represents a new strategy to fight bacterial infections. LsrK is a kinase responsible for the phosphorylation of autoinducer-2, a signaling molecule involved in quorum sensing. Inhibiting LsrK would lead to quorum sensing inactivation and interfere with the pathogenesis. In this study, we built the first LsrK 3D model and performed virtual screening of a locally available database. Selected compounds were tested against LsrK, and the analogue search conducted based on the positive hits led to the identification of low-micromolar LsrK inhibitors. These results prove the utility of the model and provide the first class of LsrK inhibitors to be further optimized as antivirulence agents.

Inhibition of Breast Cancer Resistance Protein and Multidrug Resistance Associated Protein 2 by Natural Compounds and Their Derivatives.

The food and dietary supplements we consume contain a wide variety of plant secondary metabolites and other compounds, which, like drugs, can be absorbed, metabolized, distributed, and excreted from the body. In the intestine, these compounds can interact with transport proteins such as the multidrug resistance associated protein 2 (MRP2, ABCC2) and the breast cancer resistance protein (BCRP, ABCG2) that regulate the absorption of drugs and other compounds. Inhibition of these transporters by dietary components could lead to increased exposure and adverse effects of concomitantly administered drugs. Therefore, we screened a library of 124 natural compounds and their derivatives using the vesicular transport assay to evaluate their inhibitory potential on MRP2 and BCRP. Of the library compounds, 36% were identified as BCRP inhibitors, whereas the number was only 3.2% for MRP2. BCRP inhibitors are described by higher molecular weight, number of rings, aromaticity, and LogD7.4 than noninhibitors. IC50 values were measured for six dual inhibitors, among which three novel inhibitors, gossypin, nordihydroguaiaretic acid, and octyl gallate, were identified. Our results confirm that flavonoids are avid inhibitors of BCRP, and flavones and flavonols appear to be important subclasses of flavonoids for this inhibition. The strong inhibition of BCRP transport by some compounds suggests that their presence at high levels in the diet could cause food-drug interactions, but this seems to be a minor cause of concern for MRP2.

Analyzing user-generated online content for drug discovery: development and use of MedCrawler.

Motivation: Ethnopharmacology, or the scientific validation of traditional medicine, is a respected starting point in drug discovery. Home remedies and traditional use of plants are still widespread, also in Western societies. Instead of perusing ancient pharmacopeias, we developed MedCrawler, which we used to analyze blog posts for mentions of home remedies and their applications. This method is free and accessible from the office computer. Results: We developed MedCrawler, a data mining tool for analyzing user-generated blog posts aiming to find modern 'traditional' medicine or home remedies. It searches user-generated blog posts and analyzes them for correlations between medically relevant terms. We also present examples and show that this method is capable of delivering both scientifically validated uses as well as not so well documented applications, which might serve as a starting point for follow-up research. Availability and Implementation: Source code is available on GitHub at {{ https://github.com/a-hel/medcrawler }}. Contact: paivi.tammela@helsinki.fi. Supplementary information: Supplementary data are available at Bioinformatics online.

Bioluminescent whole-cell reporter gene assays as screening tools in the identification of antimicrobial natural product extracts.

We describe novel tools, bioluminescent whole-cell reporter gene assays, for facilitating the use of natural products in antimicrobial drug discovery. As proof-of-concept, a plant extract library was screened and follow-up experiments were carried out. Primary results can be obtained in 2-4h with high sensitivity, leading to significant improvements of the process.

Integrated in vitro-in silico screening strategy for the discovery of antibacterial compounds.

Multidrug-resistant bacterial infections are an increasing source of healthcare problems, and the research for new antibiotics is currently unable to respond to this challenge. In this work, we present a screening strategy that integrates cell-based high-throughput screening (HTS) with in silico analogue search for antimicrobial small-molecule drug discovery. We performed an HTS on a diverse chemical library by using an assay based on a bioluminescent Escherichia coli K-12 (pTetLux1) strain. The HTS yielded eight hit compounds with >50% inhibition. These hits were then used for structural similarity-based virtual screening, and of the 29 analogues selected for in vitro testing, four compounds displayed potential activity in the pTetLux1 assay. The 11 most active compounds from combined HTS and analogue search were further assessed for antimicrobial activity against clinically important strains of E. coli and Staphylococcus aureus and for in vitro cytotoxicity against human cells. Three of the compounds displayed antibacterial activity and low human cell cytotoxicity. Additionally, two compounds of the set fully inhibited S. aureus growth after 24 h, but also exhibited human cell cytotoxicity in vitro.

Screening_mgmt: a Python module for managing screening data.

High-throughput screening is an established technique in drug discovery and, as such, has also found its way into academia. High-throughput screening generates a considerable amount of data, which is why specific software is used for its analysis and management. The commercially available software packages are often beyond the financial limits of small-scale academic laboratories and, furthermore, lack the flexibility to fulfill certain user-specific requirements. We have developed a Python module, screening_mgmt, which is a lightweight tool for flexible data retrieval, analysis, and storage for different screening assays in one central database. The module reads custom-made analysis scripts and plotting instructions, and it offers a graphical user interface to import, modify, and display the data in a uniform manner. During the test phase, we used this module for the management of 10,000 data points of various origins. It has provided a practical, user-friendly tool for sharing and exchanging information between researchers.

Exploring marine resources for bioactive compounds.

Biodiversity in the seas is only partly explored, although marine organisms are excellent sources for many industrial products. Through close co-operation between industrial and academic partners, it is possible to successfully collect, isolate and classify marine organisms, such as bacteria, fungi, micro- and macroalgae, cyanobacteria, and marine invertebrates from the oceans and seas globally. Extracts and purified compounds of these organisms can be studied for several therapeutically and industrially significant biological activities, including anticancer, anti-inflammatory, antiviral, antibacterial, and anticoagulant activities by applying a wide variety of screening tools, as well as for ion channel/receptor modulation and plant growth regulation. Chromatographic isolation of bioactive compounds will be followed by structural determination. Sustainable cultivation methods for promising organisms and biotechnological processes for selected compounds can be developed, as well as biosensors for monitoring the target compounds. The (semi)synthetic modification of marine-based bioactive compounds produces their new derivatives, structural analogs and mimetics that could serve as hit or lead compounds and be used to expand compound libraries based on marine natural products. The research innovations can be targeted for industrial product development in order to improve the growth and productivity of marine biotechnology. Marine research aims at a better understanding of environmentally conscious sourcing of marine biotechnology products and increased public awareness of marine biodiversity. Marine research is expected to offer novel marine-based lead compounds for industries and strengthen their product portfolios related to pharmaceutical, nutraceutical, cosmetic, agrochemical, food processing, material and biosensor applications.

Screening and characterisation of antimicrobial properties of semisynthetic betulin derivatives.

Betulin (lup-20(29)-ene-3ß, 28-diol) is a naturally occurring triterpene, which is found in substantial amounts from the outer bark of birch trees. A library of 51 structurally diverse semisynthetic betulin derivatives was screened against five bacterial strains, Enterobacter aerogenes, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus and a fungal strain Candida albicans, using broth microdilution assays. Primary antimicrobial screening at 50 µM concentration led to the identification of five compounds showing antimicrobial properties (inhibition of growth by >70% against one or more microbial strains). According to the dose-response results, 28-O-(N-acetylanthraniloyl)betulin (compound 5) was the most active, showing MIC90 of 6.25 µM against two Gram-positive bacteria, E. faecalis and S. aureus. However, the activity of this compound was affected by albumin binding, which was demonstrated by the loss of activity in a host-pathogen co-culture assay as well as in the antibacterial assay in the presence of increased concentration of albumin. Furthermore, the effects on mammalian cells were evaluated by cytotoxicity assessment on hepatocyte cell culture after 24 h exposure to the compounds. Betulinic aldehyde (18), betulin-28-oxime (31) and hetero cycloadduct with acetoxy groups at carbon atoms 3 and 28 and ethyl substituent at the triazolo ring (43) displayed cytotoxicity towards hepatocytes, with IC50 values of 47, 25 and 16 µM, respectively. The IC50 value for 28-O-(N-acetylanthraniloyl)betulin (5) was 56 µM. The current study presents an insight into using betulin scaffold for developing derivatives with antibacterial potential, and furthermore the necessity of in-depth analysis of found actives through selectivity profiling and follow-up studies including in silico ADMET predictions.

Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays.

Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC50 values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.

Triterpenoid acids and lactones from the leaves of Fadogia tetraquetra var. tetraquetra (Rubiaceae).

Four triterpenoids isolated from the leaves of Fadogia tetraquetra var. tetraquetra, 3beta-hydroxy-11alpha, 12alpha-epoxyoleanan-28,13beta-olide (1), 3beta-hydroxyurs-11-en-28,13beta-olide (2), oleanolic acid (3), and ursolic acid (4), were evaluated for their antiviral and antibacterial properties. Compound 4 showed potent activity against the Semliki Forest virus with an IC50 of 14.7 microM, but was also found to be significantly cytotoxic (68% reduction in cell viability after 24 hours exposure at 50 microM) towards baby hamster kidney (BHK21) host cells. A viability assay on the mammalian human hepatocellular carcinoma (Huh-7) cell line showed no significant effects on intracellular ATP content after 48 hours exposure to compounds 1-4 at this concentration. Compound 4 also inhibited Staphylococcus aureus (MIC 12.5 microM), but was inactive against Enterobacter aerogenes, Escherichia coli, and Pseudomonas aeruginosa. Compounds 1-3 were inactive against all tested bacterial strains at 50 microM concentration.

Discovering protein kinase C active plants growing in Finland utilizing automated bioassay combined to LC/MS.

Protein kinase C (PKC) modulating activity of 81 plant extracts of Finnish origin was investigated with an automated bioassay method combined to LC/MS. Twenty-one extracts from different parts of the plants inhibited PKC significantly. Fractionation of the active extract of Filipendula ulmaria showed that this method was able to identify a PKC inhibiting compound from the extract as quercetin. Our results indicate that this method is suitable for PKC screening of complex matrices and provides a quick and low volume, non-radioactive, alternative method for PKC experiments.

Comparison of transgenic Gerbera hybrida lines and traditional varieties shows no differences in cytotoxicity or metabolic fingerprints.

Genetic modification using gene transfer (GM) is still controversial when applied to plant breeding at least in Europe. One major concern is how GM affects other genes and thus the metabolism of the plant. In this study, 225 genetically modified lines of the ornamental plant Gerbera hybrida and 42 non-GM gerbera varieties were used to investigate changes in secondary metabolism. The cytotoxicity of GM and non-GM gerbera extracts was evaluated on human cell lines derived from lung, liver, and intestinal tissues. The results indicate that the safety profile for GM gerbera lines is similar to the viability pattern for non-GM varieties-none of the extracts were toxic. In addition, metabolic fingerprints of gerbera extracts were identified using thin-layer chromatography and analysed by principal component analysis (PCA), the nearest neighbour classifier, and Fligner-Killeen test. No new compounds unique to GM lines were observed. With PCA, no separation between GM gerbera lines and varieties could be demonstrated. In the nearest neighbour classifier, 54% of the samples found the expected neighbour based on the gene constructs used for transformation. With Fligner-Killeen test, we studied if the amounts of compounds vary more in GM gerberas than in varieties. In most cases, there were no statistically significant differences between the varieties and GM lines or there was more variation among the non-GM varieties than in the GM lines. The variance of a single compound was significantly larger in transgenic gerbera lines than in varieties and of three compounds in non-GM varieties.

Assessing the data quality in predictive toxicology using a panel of cell lines and cytotoxicity assays.

In vitro cell viability assays have a central role in predictive toxicology, both in assessing acute toxicity of chemicals and as a source of experimental data for in silico methods. However, the quality of in vitro toxicity databanks fluctuates dramatically because information they contain is obtained under varying conditions and in different laboratories. The aim of this study was to identify the factors responsible for these deviations and thus the quality of the data extracted for predictive toxicology. Three cell viability assays measuring LDH leakage, WST-1 reduction, and intracellular ATP were compared in an automated environment using four mammalian cell lines: Caco-2, Calu-3, Huh-7, and BHK. Using four standard compounds--polymyxin B, gramicidin, 5-fluorouracil, and camptothecin--a significant lack of sensitivity in LDH assay compared with the other assays was observed. Because the viability IC(50) values for the standards were similar among the cell lines, the biochemical characteristics of different cell lines seem to play only a minor role, with an exception being the hepatocellular Huh-7 cell line. Toxicity assessment of new 1,2,4-triazoles revealed significant differences in their toxic potential, and the results indicate the same sensitivity profile among the assays as observed with the standard compounds. Overall, it can be argued that the assay selection is the most important factor governing the uniform quality of the data obtained from in vitro cell viability assays.

In-vitro mutagenic potential and effect on permeability of co-administered drugs across Caco-2 cell monolayers of Rubus idaeus and its fortified fractions.

This study investigated the mutagenic, anti-mutagenic and cytotoxic effects of acetone extract of raspberry, Rubus idaeus L. (v. Ottawa) Rosaceae, and the isolated and characterized ellagitannin and anthocyanin fractions thereof, suitable for food applications. The studied raspberry extract and fractions did not show any mutagenic effects determined in the miniaturized Ames test and were not cytotoxic to Caco-2 cells at the used concentrations. However, the anti-mutagenic properties were changed (i.e. decreased mutagenicity of 2-nitrofluorene in strain TA98, and slightly increased mutagenicity of 2-aminoanthracene in strain TA100) with metabolic activation. Further, their influence on the permeability of co-administered common drugs (ketoprofen, paracetamol, metoprolol and verapamil) across Caco-2 monolayers was evaluated. The apical-to-basolateral permeability of highly permeable verapamil was mostly affected (decreased) during co-administration of the raspberry extract or the ellagitannin fraction. Ketoprofen permeability was decreased by the ellagitannin fraction. Consumption of food rich in phytochemicals, as demonstrated here with chemically characterized raspberry extract and fractions, with well-absorbing drugs would seem to affect the permeability of some of these drugs depending on the components. Thus their effects on the absorption of drugs in-vivo cannot be excluded.

Effects of extracts of commonly consumed food supplements and food fractions on the permeability of drugs across Caco-2 cell monolayers.

PURPOSE: Extracts made from berries, herbs, and various plant materials, which might possess a range of activities, are used as health promoting products. Because little is known about their effects on the absorption of co-administered drugs, the effects of some food supplements, Finnish berries, and herbs were studied on the permeability of some commonly used drugs. METHODS: The permeabilities of verapamil, metoprolol, ketoprofen, paracetamol, and furosemide were studied across Caco-2 cell monolayers with contemporaneously administered extracts from flax seed, purple loosestrife, and Scots pine bark; bilberries, cowberries, and raspberries; oregano, rosemary, and sage. Toxicological tests were conducted to determine cellular damage. RESULTS: The effects of extracts on drug permeabilities were generally minor. Flax seed decreased the permeability of all drugs except verapamil. Purple loosestrife and pine decreased verapamil and metoprolol permeability. Changes caused by berries were mainly pH-related. Rosemary and oregano enhanced furosemide permeability. CONCLUSIONS: Ingestion of extracts of herbs and berries studied are not expected to markedly change the permeabilities of highly permeable drugs. Harmful effects at sites of or during absorption are unlikely. However, if high doses of extracts are administered with low permeable drugs in vitro, effects on drug permeabilities could not be excluded. Use of such extracts should therefore be evaluated during continuous medication.

HPLC micro-fractionation coupled to a cell-based assay for automated on-line primary screening of calcium antagonistic components in plant extracts.

High performance liquid chromatography (HPLC) micro-fractionation was successfully coupled to an automated 45Ca2+ uptake assay using GH4C1 cells for the separation of natural product extracts and for the primary detection of their calcium antagonistic components. The reliability of the procedure was first established with a reference solution consisting of pure compounds with a known effect on the Ca2+ uptake. No loss of activity was observed to occur after HPLC micro-fractionation. Extracts of Peucedanum palustre and Pinus sylvestris, showing high and no inhibition of Ca2+ uptake as total extracts, respectively, were analysed and the inhibitory activity of the P. palustre extract could be traced to two components, identified as columbianadin and isoimperatorin. As expected, no significant inhibition was observed with the micro-fractionated P. sylvestris samples. In summary, the procedure was found to be applicable for primary detection of calcium antagonistic components in complex matrices and to significantly reduce the time previously needed for bioactivity-guided isolation.