Quercetin is a flavonoid compound widely present in plants and exhibits a variety of biological activities. Research on quercetin has shown its potential for medical application. In this research, we elucidate its antioxidant mechanism and the broad-spectrum antibacterial and antiparasite properties; summarise its potential application in antioncology and cardiovascular protection and anti-immunosuppression treatment; and demonstrate its ability to alleviate the toxicity of mycotoxins. This research is expected to offer some insights and inspirations for the further study of quercetin, its properties, and the scientific basis for its better application in clinical practice.

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Quercetin: Its Main Pharmacological Activity and Potential Application in Clinical Medicine.

The microtiter assay is one of the most widely used methods for assessing biofilm formation. Though it has high throughput, this assay is known for its substantial deviation from experiment to experiment, and even from well to well. Since the assay constitutes one of the pillars of biofilm research, it was decided to examine the wells of a microtiter plate directly during growth, treatment, and the steps involved in crystal violet (CV) measurements. An inverted Zeiss LSM 880 confocal laser scanning microscope was used to visualize and quantify biomass directly in the wells of the microtiter plate. Green fluorescent protein-tagged Pseudomonas aeruginosa, PAO1, and live/dead stains were used to assess the structure, state, and position of biomass build-up. Microscopic observations were compared with colony-forming unit (CFU) and CV measurements. The development and the structured architecture of biomass was observed in real-time in the wells. Three-dimensional images of biomass were obtained from all of the microtiter wells; these showed variations from well to well. CV staining showed large variations in remaining biomass, depending on the method selected to remove the supernatant prior to CV staining (i.e. pipetting or manually discarding the fluid by inversion, washed or unwashed wells). Colony-forming unit counts or live/dead staining used to evaluate biomass with or without antibiotic treatment proved imprecise due to aggregation, limited removal of biomass, and overestimation of dead staining. The highly structured microenvironment of biomass in microtiter wells needs to be considered when designing and analyzing experiments. When using microtiter plates, stochastic variation due to growth and handling may lead to flawed conclusions. It is therefore recommended that this assay be used as a screening tool rather than as a stand-alone experimental tool.

Into the well-A close look at the complex structures of a microtiter biofilm and the crystal violet assay.

Quercetin, an essential plant flavonoid, possesses a variety of pharmacological activities. Extensive literature investigates its antimicrobial activity and possible mechanism of action. Quercetin has been shown to inhibit the growth of different Gram-positive and Gram-negative bacteria as well as fungi and viruses. The mechanism of its antimicrobial action includes cell membrane damage, change of membrane permeability, inhibition of synthesis of nucleic acids and proteins, reduction of expression of virulence factors, mitochondrial dysfunction, and preventing biofilm formation. Quercetin has also been shown to inhibit the growth of various drug-resistant microorganisms, thereby suggesting its use as a potent antimicrobial agent against drug-resistant strains. Furthermore, certain structural modifications of quercetin have sometimes been shown to enhance its antimicrobial activity compared to that of the parent molecule. In this review, we have summarized the antimicrobial activity of quercetin with a special focus on its mechanistic principle. Therefore, this review will provide further insights into the scientific understanding of quercetin’s mechanism of action, and the implications for its use as a clinically relevant antimicrobial agent.

https://www.mdpi.com/1420-3049/27/8/2494/pdf?version=1649767293

Antimicrobial Activity of Quercetin: An Approach to Its Mechanistic Principle

Bacterial biofilms are multicellular aggregates enclosed in a self-created biopolymer matrix. Biofilm-producing bacteria have become a great public health problem worldwide because biofilms enable these microorganisms to evade several clearance mechanisms produced by host and synthetic sources. Over the past years, different flavonoids including quercetin have engrossed considerable interest among researchers owing to their potential anti-biofilm properties. To our knowledge, there is no review regarding effects of quercetin towards bacterial biofilms, prompting us to summarize experimental evidence on its anti-biofilm properties. Quercetin inhibits biofilm development by a diverse array of bacterial pathogens such as Enterococcus faecalis, Staphylococcus aureus, Streptococcus mutans, Escherichia coli, and Pseudomonas aeruginosa. Prevention of bacterial adhesion, suppression of quorum-sensing pathways, disruption or alteration of plasma membrane, inhibition of efflux pumps, and blocking nucleic acid synthesis have been documented as major anti-biofilm mechanisms of quercetin. Overall, anti-biofilm activity of quercetin can open up new horizons in a wide range of biomedical areas, from food industry to medicine.

An overview on anti-biofilm properties of quercetin against bacterial pathogens

Helicobacter pylori is a bacterium known mainly of its ability to cause persistent inflammations of the human stomach, resulting in peptic ulcer diseases and gastric cancers. Continuous exposure of this bacterium to antibiotics has resulted in high detection of multidrug-resistant strains and difficulties in obtaining a therapeutic effect. The purpose of the present study was to determine the usability of bacterial cellulose (BC) chemisorbed with 3-bromopyruvate (3-BP) or sertraline (SER) to act against lawn H. pylori biofilms. The characterization of BC carriers was made using a N2 adsorption/desorption analysis, tensile strength test, and scanning electron microscopy (SEM) observations. Determination of an antimicrobial activity was performed using a modified disk-diffusion method and a self-designed method of testing antibacterial activity against biofilm microbial forms. In addition, bacterial morphology was checked by SEM. It was found that BC disks were characterized by a high cross-linking and shear/stretch resistance. Growth inhibition zones for BC disks chemisorbed with 2 mg of SER or 3-BP were equal to 26.5-27.5 mm and 27-30 mm, respectively. The viability of lawn biofilm H. pylori cells after a 4-h incubation with 2 mg SER or 3-BP chemisorbed on BC disks was ≥4 log lower, suggesting their antibacterial effect. SEM observations showed a number of morphostructural changes in H. pylori cells exposed to these substances. Concluding, SER and 3-BP chemisorbed on BC carriers presented a promising antibacterial activity against biofilm H. pylori cells in in vitro conditions.

https://www.mdpi.com/1422-0067/21/24/9507/pdf

Potential of Bacterial Cellulose Chemisorbed with Anti-Metabolites, 3-Bromopyruvate or Sertraline, to Fight against Helicobacter pylori Lawn Biofilm.

Microtiter plate-based bacterial biofilm assay is frequently used to study bacterial biofilm development and growth While this assay is simple and relatively high-throughput, it frequently shows difficulty in establishing robust biofilm attachment in the wells We report that the consistency of bacterial biofilm assays carried out in microtiter plates subjected to abrasive treatment, by sandblasting or drill press grinding, is significantly improved in a Pseudomonas fluorescens Pf0-1 model Scanning electron microscopy imaging suggests that the treated surfaces could provide points of attachment to facilitate the recruitment of bacteria in the initial phase of biofilm colony establishment The sandblast treated polypropylene, but not polystyrene, plates were found suitable in studying the impact of flavonoid quercetin on the biofilm formation in Bacillus subtilis FB17 Further investigation revealed that due to the hydrophobicity of the polystyrene surfaces, a greater amount of quercetin was adsorbed on the plate surface, effectively lowering the concentration of the flavonoid in solution

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Abrasive treatment of microtiter plates improves the reproducibility of bacterial biofilm assays

Four 4-aryl-N-methoxyethyl-1,8-naphthalimide derivatives were synthesized and evaluated for their fluorescent properties. All four compounds, 4-(p-methoxy)- 3a, 4-(p-amino)- 3b, 4-(p-dimethylamino)- 3c, and 4-(p-acetamido)- 3d are highly fluorescent in chloroform, but their fluorescence intensity is drastically decreased in polar solvents such as DMSO and methanol. Derivative 3c, in particular, showed a 699-fold fluorescence quenching in methanol, as compared in chloroform at 10 μM dye concentration. Furthermore, dilution of DMSO solutions of probes 3a, 3b and 3d with water led to drastic decrease in fluorescence intensity, and virtually complete fluorescence quenching in the case of 3d. In aqueous solutions containing 3a or 3b, the presence of bovine serum albumin led to 80- and 50-fold increases in fluorescence intensity, respectively.

1,8-Naphthalimide derivatives as probes for protein surface hydrophobicity

Using 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene as starting material, N,N,N-trimethyl-2-(4,4-difluoro-4-bora-3a,4a-diaza-s-indacen-8-yl)ethylammonium iodide (BODIPY quaternary ammonium 17) was successfully synthesized. While this BODIPY ammonium was found to be less stable than Rose Bengal both in light and dark, it showed a superior efficiency as a photosensitizer, leading to the photodegradation of 1,3-diphenylisobenzofuran at a rate about 2.6 times faster than Rose Bengal. Supplementation of this BODIPY in agar cultures did not show antibacterial activity against Escherichia coli DE3 exposed to light, however, inclusion of 5 μM BODIPY 17 in agar plates led to complete killing to Bacillus subtilis while exposed to light. 

BODIPY quaternary ammonium salt as photosensitizers

Denaturing anion exchange HPLC simplifies the chromatographic profiles of self-complementary sequences such as d(CG)6, d(CG)6 with locked nucleic acid modifications, d(AT)15, and polymerase chain reaction mixtures. These chromatographic conditions use eluents containing up to 4 M urea at pH 12.4, and lead to the abolishment of secondary structures and meaningful chromatographic patterns of self-complementary sequences. Similarly, PCR template, FAM-labelled primer and FAM-labelled PCR products were resolved, making interpretation of PCR reaction products possible.

Anion exchange chromatography of oligonucleotides under denaturing conditions

4-Phenyl-1,8-naphthalimide was synthesized by imidation of commercially available 4-bromo-1,8-naphthalic anhydride, followed by Suzuki coupling with phenyl boronic acid, both under microwave heating. The microwave-assisted reactions were found to be faster and more efficient than reactions carried out by heating in oil-baths. While this compound was quite fluorescent in solvents such as chloroform, methanol and ethanol, it is virtually non-fluorescent in DMSO; however, upon the addition of water to DMSO solutions of this dye, fluorescence was restored, suggesting a tendency for aggregation-induced emission. The fluorescent properties of 4-phenyl-1,8-naphthalimide were found to be influenced by salt concentrations, likely as a result of hydrophobic effects. While this dye does not show binding to DNA, presence of bovine serum albumin leads to effective fluorescence quenching.

https://www.mdpi.com/1422-8599/2020/2/M1127/pdf

Frank Betancourt

Papers

Abrasive treatment of microtiter plates improves the reproducibility of bacterial biofilm assays

1,8-Naphthalimide derivatives as probes for protein surface hydrophobicity

BODIPY quaternary ammonium salt as photosensitizers

Anion exchange chromatography of oligonucleotides under denaturing conditions

Microwave-Assisted Synthesis and Fluorescent Properties of 4-Phenyl-1,8-naphthalimide