Pia Vuorela

Anti-Inflammatory Effects of Flavonoids: Genistein, Kaempferol, Quercetin, and Daidzein Inhibit STAT-1 and NF-κB Activations, Whereas Flavone, Isorhamnetin, Naringenin, and Pelargonidin Inhibit only NF-κB Activation along with Their Inhibitory Effect on iNOS Expression and NO Production in Activated Macrophages

In inflammation, bacterial products and proinflammatory cytokines induce the formation of large amounts of nitric oxide (NO) by inducible nitric oxide synthase (iNOS), and compounds that inhibit NO production have anti-inflammatory effects. In the present study, we systematically investigated the effects of 36 naturally occurring flavonoids and related compounds on NO production in macrophages exposed to an inflammatory stimulus (lipopolysaccharide, LPS), and evaluated the mechanisms of action of the effective compounds. Flavone, the isoflavones daidzein and genistein, the flavonols isorhamnetin, kaempferol and quercetin, the flavanone naringenin, and the anthocyanin pelargonidin inhibited iNOS protein and mRNA expression and also NO production in a dose-dependent manner. All eight active compounds inhibited the activation of nuclear factor-κB (NF-κB), which is a significant transcription factor for iNOS. Genistein, kaempferol, quercetin, and daidzein also inhibited the activation of the signal transducer and activator of transcription 1 (STAT-1), another important transcription factor for iNOS. The present study characterises the effects and mechanisms of naturally occurring phenolic compounds on iNOS expression and NO production in activated macrophages. The results partially explain the pharmacological efficacy of flavonoids as anti-inflammatory compounds.

Binding of Phenolic Compounds and Their Derivatives to Bovine and Reindeer β-Lactoglobulin

In plant-based food, phenolic compounds usually do not exist in their native form, but as esters, glycosides, or polymers. The native forms, however, require deglycosylation for their intestinal absorption, and aglycone has been considered to be the potential health-protecting/promoting form. The binding of the aglycones of phenolic compounds to bovine and reindeer beta-lactoglobulins (betaLG) using fluorescence quenching was studied. The effects of pH and storage were also studied. Of the compounds investigated, the majority of flavones, flavonols, flavanones, and isoflavones were bound to betaLG. In the pH studies, no significant effects were found. The fact that the phenolic compounds were not released at pH 2 might indicate that they bind to an external part rather than to the central cavity. Studies implicated that betaLG could act as a binder or carrier for phenolic compounds in acidic, basic, or neutral conditions and that the ligand/betaLG complex can remain stable during storage.

Towards fabrication of 3D printed medical devices to prevent biofilm formation

The use of three-dimensional (3D) printing technologies is transforming the way that materials are turned into functional devices. We demonstrate in the current study the incorporation of anti-microbial nitrofurantoin in a polymer carrier material and subsequent 3D printing of a model structure, which resulted in an inhibition of biofilm colonization. The approach taken is very promising and can open up new avenues to manufacture functional medical devices in the future.

Tailoring controlled-release oral dosage forms by combining inkjet and flexographic printing techniques.

We combined conventional inkjet printing technology with flexographic printing to fabricate drug delivery systems with accurate doses and tailored drug release. Riboflavin sodium phosphate (RSP) and propranolol hydrochloride (PH) were used as water-soluble model drugs. Three different paper substrates: A (uncoated woodfree paper), B (triple-coated inkjet paper) and C (double-coated sheet fed offset paper) were used as porous model carriers for drug delivery. Active pharmaceutical ingredient (API) containing solutions were printed onto 1 cm × 1 cm substrate areas using an inkjet printer. The printed APIs were coated with water insoluble polymeric films of different thickness using flexographic printing. All substrates were characterized with respect to wettability, surface roughness, air permeability, and cell toxicity. In addition, content uniformity and release profiles of the produced solid dosage forms before and after coating were studied. The substrates were nontoxic for the human cell line assayed. Substrate B was smoothest and least porous. The properties of substrates B and C were similar, whereas those of substrate A differed significantly from those of B, C. The release kinetics of both printed APIs was slowest from substrate B before and after coating with the water insoluble polymer film, following by substrate C, whereas substrate A showed the fastest release. The release rate decreased with increasing polymer coating film thickness. The printed solid dosage forms showed excellent content uniformity. So, combining the two printing technologies allowed fabricating controlled-release oral dosage forms that are challenging to produce using a single technique. The approach opens up new perspectives in the manufacture of flexible doses and tailored drug-delivery systems.

Effects of Flavonoids on Prostaglandin E2 Production and on COX-2 and mPGES-1 Expressions in Activated Macrophages

Prostaglandin E2 (PGE2) has a central role in inflammation and both cyclooxygenase-2 (COX-2) and prostaglandin E synthases are critical enzymes in its synthesis. In inflammation, bacterial products and cytokines enhance the expression of COX-2 and inducible microsomal prostaglandin E synthase-1 (mPGES-1) which are functionally coupled to result in increased PGE2 formation in macrophages and tissue cells. In the present study, we systematically investigated the effects of 26 naturally occurring flavonoids on PGE2 production and on COX-2 and mPGES-1 expression in activated macrophages. Twelve flavonoids, i.e., flavone, luteolin-7-glucoside, kaempferol, isorhamnetin, morin, quercetin, naringenin, taxifolin, pelargonidin, daidzein, genistein, and genistin effectively inhibited lipopolysaccharide (LPS)-induced PGE2 production. Four flavonoids (flavone, isorhamnetin, daidzein, and genistein) inhibited significantly LPS-induced COX-2 expression, while mPGES-1 expression was downregulated by kaempferol and isorhamnetin. The present study characterizes the effects of flavonoids on PGE2 production and on COX-2 and mPGES-1 expression in activated macrophages. The results add to our knowledge of the anti-inflammatory actions of flavonoids and introduce kaempferol and isorhamnetin as compounds capable of downregulating the expression of mPGES-1.

Inhibition of acetylcholinesterase by coumarins: The case of coumarin 106

In this contribution, from a coumarin library consisting of 29 compounds including natural and synthetic derivatives, an active acetylcholinesterase (AChE) inhibitor (coumarin 106) was found. This circumstance leaded us to continue with the pharmacological characterization of coumarin 106. The first study with the coumarin library was performed using a 96-microtiter well plate assay based on Ellmans reaction. Coumarins were assayed at 5 and 30 microM, and coumarin 106 was found the most active inhibitor at both concentrations. The follow-up analysis using kinetic studies demonstrated that coumarin 106 displays mixed-type AChE inhibition with a pIC(50)=4.97+/-0.09 and K(i)=2.36+/-0.17 microM. The ability of this molecule to interact with AChE was further confirmed through computational studies, in which a primary binding was proved to occur at the active gorge site, while a secondary binding was demonstrated at the peripheral anionic site. Also, coumarin 106 was shown to inhibit butyrylcholinesterase (BChE) with slightly lower potency (pIC(50)=4.56+/-0.06), and found to be non-toxic in Caco-2 cells. The combination of these findings makes coumarin 106 an attractive molecule for further investigation. This is the first report where AChE inhibitory activity has been associated with coumarin 106, and proof has been given of its convenience as a lead molecule.

The chemical and biological activities of quinones: overview and implications in analytical detection

Quinones are a class of natural and synthetic compounds that have several beneficial effects. Quinones are electron carriers playing a role in photosynthesis. As vitamins, they represent a class of molecules preventing and treating several illnesses such as osteoporosis and cardiovascular diseases. Quinones, by their antioxidant activity, improve general health conditions. Many of the drugs clinically approved or still in clinical trials against cancer are quinone related compounds. Quinones have also toxicological effects through their presence as photoproducts from air pollutants. Quinones are fast redox cycling molecules and have the potential to bind to thiol, amine and hydroxyl groups. The aforementioned properties make the analytical detection of quinones problematic. However, recent advances of the available analytical techniques along with the possibility of using labeled compound facilitate their detection hence allowing a better understanding of their action. This review summarizes the current knowledge with respect to the oxido-reductive and electrophilic properties of quinones as well as to the analytical tools used for their analysis. It includes a general introduction about the physiological, and therapeutical functions of quinones. A number of studies are reported to cover the chemical reactivity in an attempt to understand quinones as biologically active compounds. Data ranging from normal analytical methods to study quinones derived from plant or biological matrices to the use of labeled compounds are presented. The examples illustrate how chemical, biological and analytical knowledge can be integrated to have a better understanding of the mode of action of the quinones.

Pros and cons of using resazurin staining for quantification of viable Staphylococcus aureus biofilms in a screening assay

Staining of Staphylococcus aureus biofilms with 20 microM resazurin during 20 min was shown to provide a good screening assay in 96-well micro titer plates. However, data quality was found to be dependent on the staining duration and biofilm concentration. Also, the inadequacy of using resazurin calibration curves with planktonic cells to estimate S. aureus biofilm concentrations was demonstrated.

Automating a 96-well microtitre plate model for Staphylococcus aureus biofilms : an approach to screening of natural antimicrobial compounds

The purpose of this study was to establish and automate an assay to be used for screening novel antimicrobial agents against biofilm-forming Staphylococcus aureus bacteria. The selected assay was based on crystal violet staining, which is a well used method for staining bacterial biofilms. The method was first optimised manually, antibiotic susceptibility was established and biofilm formation in plates was confirmed using atomic force microscopy. Automation of the assay was done using a Thermo Scientific Multidrop((R)) Combi dispenser and Biomek((R)) 3000 liquid handling workstation. A detailed comparison of the performance between the manual and the automated method was made in terms of screening window coefficient as well as other statistical parameters and repeatability measurements, such as plate-to-plate and day-to-day variability. Automated screening of an in-house library of natural products gave the same positive hits as previously reported, therefore the developed assay can be regarded as a reliable screening tool.

Combining biofilm matrix measurements with biomass and viability assays in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms

Despite that three types of assays (measuring biofilm viability, biomass, or matrix) are described to assess anti-biofilm activity, they are rarely used together. As infections can easily reappear if the matrix is not affected after antibiotic treatments, our goal was to explore the simultaneous effects of antibiotics on the viability, biomass and matrix of Staphylococcus aureus biofilms (ATCC 25923). Viability and biomass were quantified using resazurin and crystal violet staining sequentially in the same plate, while matrix staining was conducted with a wheat germ agglutinin-Alexa Fluor 488 fluorescent conjugate. Establishment of the detection limits and linearity ranges allowed concluding that all three methods were able to estimate biofilm formation in a similar fashion. In a susceptibility study with 18-h biofilms, two model compounds (penicillin G and ciprofloxacin) caused a reduction on the viability and biomass accompanied by an increase or not changed levels of the matrix, respectively. This response pattern was also proven for S. aureus Newman, S. epidermidis and E. coli biofilms. A classification of antibiotics based on five categories according to their effects on viability and matrix has been proposed earlier. Our data suggests a sixth group, represented by penicillin, causing decrease in bacterial viability but showing stimulatory effects on the matrix. Further, if effects on the matrix are not taken into account, the long-term chemotherapeutic effect of antibiotics can be jeopardized in spite of the positive effects on biofilms viability and biomass. Thus, measuring all these three endpoints simultaneously provide a more complete and accurate picture.