We put forward an efficient strategy based on bioassay guidance for the rapid screening, identification, and purification of the neuraminidase inhibitors from traditional Chinese medicines, and apply ...to the discovery of anti‐influenza components from Lithospermiun erythrorhizon Sieb.et Zucc. Ultrafiltration with high‐performance liquid chromatography and electrospray ionization time‐of‐flight mass spectrometry was employed for the rapid screening and preliminarily identification of anti‐influenza components from Zicao. Semipreparative high‐performance liquid chromatography was used for the rapid separation and purification of the target compounds. NMR spectroscopy, mass spectrometry, and UV spectroscopy were used for further structural identification, and the activity of the compounds was verified by in vitro assay. Five compounds were found to have neuraminidase inhibitory activity by this method. Subsequently, the five compounds were separated by semipreparative high‐performance liquid chromatography with the purity over 98% for all of them by high‐performance liquid chromatography test. Combined with the NMR spectroscopy, mass spectrometry, and UV spectroscopy data, they were identified as alkannin, acetylalkannin, isobutyrylalkannin, β,β‐dimethylacryloylalkannin and isovalerylalkannin. The in vitro assay showed that all five compounds had good neuraminidase inhibitory activities. These results suggested that the method is highly efficient, and it can provide platform and methodology supports for the rapid discovery of anti‐influenza active ingredients from complex Chinese herbal medicines.
Prodigiosin, a red linear tripyrrole pigment, has long been recognised for its antimicrobial property. However, the physiological contribution of prodigiosin to the survival of its producing hosts ...still remains undefined. Hence, the aim of this study was to investigate the biological role of prodigiosin from Serratia marcescens, particularly in microbial competition through its antimicrobial activity, towards the growth and secreted virulence factors of four clinical pathogenic bacteria (methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa) as well as Staphylococcus aureus and Escherichia coli. Prodigiosin was first extracted from S. marcescens and its purity confirmed by absorption spectrum, high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrophotometry (LC-MS/MS). The extracted prodigiosin was antagonistic towards all the tested bacteria. A disc-diffusion assay showed that prodigiosin is more selective towards Gram-positive bacteria and inhibited the growth of MRSA, S. aureus and E. faecalis and Gram-negative E. coli. A minimum inhibitory concentration of 10 mug/muL of prodigiosin was required to inhibit the growth of S. aureus, E. coli and E. faecalis whereas > 10 mug/muL was required to inhibit MRSA growth. We further assessed the effect of prodigiosin towards bacterial virulence factors such as haemolysin and production of protease as well as on biofilm formation. Prodigiosin did not inhibit haemolysis activity of clinically associated bacteria but was able to reduce protease activity for MRSA, E. coli and E. faecalis as well as decrease E. faecalis, Salmonella Typhimurium and E. coli biofilm formation. Results of this study show that in addition to its role in inhibiting bacterial growth, prodigiosin also inhibits the bacterial virulence factor protease production and biofilm formation, two strategies employed by bacteria in response to microbial competition. As clinical pathogens were more resistant to prodigiosin, we propose that prodigiosin is physiologically important for S. marcescens to compete against other bacteria in its natural soil and surface water environments.
Polysorbate 80 (PS80) is widely used as an excipient in vaccines and biopharmaceuticals. The oxidized species of PS80 have raised concern because of their potential to compromise product stability ...and pose a clinical risk. Analytical methods to profile and identify the oxidized species are hard to develop owing to their complexity and low abundance. Herein, a novel strategy was demonstrated to comprehensively profile and identify the oxidized species of PS80 using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The characteristic fragmentation patterns of the oxidized species were obtained under the “all ions” scan mode. Then, 10 types of distinct fragments from oxidized oleates were identified and confirmed using two purified oxidized species (polyoxyethylene (POE) sorbitan mono-hydroxy oleate and POE mono-keto oleate) whose structures were elucidated via nuclear magnetic resonance. A total of 348 (32 types) oxidized species were profiled and identified in the oxidized PS80 samples, including 119 (10 types) species found for the first time to our knowledge. Mathematical models were established and validated based on the good logarithmic relation between the POE degree of polymerization and the relative retention time and used to rapidly discover and identify the oxidized species. A novel strategy was established to profile and identify the PS80 oxidized species based on their retention time, HRMS, and HRMS2 data of the detected peaks using an in-house dataset. Using this strategy, 104 (14 types) and 97 (13 types) oxidized species were identified for the first time in PS80 and its preparations, respectively.
In this study, an advanced ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was developed for quantifying ethoxyquin (EQ). The approach employed a ...distinctive antioxidant added extraction step designed to prevent ethoxyquin decomposition and maintain analytical precision. This method effectively determines residue levels of EQ in eggs, processed egg products, poultry muscle, salmon, and liquid milk. The method was shown to have a limit of quantitation (LOQ) for eggs, milk, salmon, and chicken muscle of 1.5 µg/kg, 1.9 µg/kg, 2.1 µg/kg, and 1.2 µg/kg, respectively. The recoveries of EQ ranged from 79.2% to 107.6%, with a relative standard deviation (RSD) below 8.4%. A surveillance study for the presence of EQ in different types of eggs and poultry muscle available in Singapore was conducted and a total of 140 samples were tested. EQ residues in all samples were found to be below the U.S. Food and Drug Administration (FDA) MRLs of 500 µg/kg. Some samples of salted and preserved eggs from China were detected with higher concentration of EQ.
Quorum sensing is a mechanism of bacterial communication that controls virulence gene expression. Pseudomonas aeruginosa regulates virulence via two synthase/transcription factor receptor pairs: ...LasI/R and RhlI/R. LasR is considered the master transcriptional regulator of quorum sensing, as it upregulates rhlI/R. However, clinical isolates often have inactivating mutations in lasR, while maintaining Rhl-dependent signaling. We sought to understand how quorum sensing progresses in isolates with lasR mutations, specifically via activation of RhlR. We find that clinical isolates with lasR inactivating mutations often harbor concurrent mutations in rhlI. Using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry, we discover that strains lacking lasR overproduce the RhlI-synthesized autoinducer and that RhlI variants re-calibrate autoinducer concentrations to wild-type levels, restoring virulent phenotypes. These findings provide a mechanism for the plasticity of quorum sensing progression in an acute infection niche.
To harness the synthetic power of modular polyketide synthases (PKSs), many aspects of their biochemistry must be elucidated. A robust platform to study these megadalton assembly lines has not yet ...been described. Here, we in vitro reconstitute the venemycin PKS, a short assembly line that generates an aromatic product. Incubating its polypeptides, VemG and VemH, with 3,5-dihydroxybenzoic acid, ATP, malonate, coenzyme A, and the malonyl-CoA ligase MatB, venemycin production can be monitored by HPLC and NMR. Multi-milligram quantities of venemycin are isolable from dialysis-based reactors without chromatography, and the enzymes can be recycled. Assembly line engineering is performed using pikromycin modules, with synthases designed using the updated module boundaries outperforming those using the traditional module boundaries by over an order of magnitude. Using combinations of VemG, VemH, and their engineered derivatives, as well as the alternate starter unit 3-hydroxybenzoic acid, a combinatorial library of six polyketide products is readily accessed.
•A novel MIECS was constructed for OLA detection.•Dopamine@graphene composites and PPy were explored for enlarging the MIECS signal.•The MIECS used two properties of dopamine: adhesion and functional ...monomers.•The developed sensor displayed high sensitivity, good selectivity, reproducibility.
In this paper, an advanced molecularly imprinted electrochemical sensor (MIECS) based on electropolymerized olaquindox (OLA) surface molecularly imprinted polymer thin film on a modified glassy carbon electrode (GCE) was developed for the detection of OLA. It was fabricated by coating dopamine@graphene (DGr) on GCE, then electropolymerizing pyrrole (Py) and molecularly imprinted polymers (MIPs). Graphene (Gr) was introduced for improving conductivity and sensitivity. Dopamine (DA) was used for dispersion and adhesion of Gr. Polypyrrole (PPy) could fix DGr and enhance the current response evidently. The established sensor could selectively recognize OLA but not the analogs of OLA. Some essential parameters controlling the performance of the developed sensor were investigated and optimized. Under optimal conditions, the linear relationship between the current intensity and OLA concentration was obtained from 50 nmol L−1 to 500 nmol L−1 with a limit of detection (LOD) of 7.5 nmol L−1. Analytical results of OLA based on the developed MIECS for fish and feedstuffs showed a good agreement with the results based on high performance liquid chromatography (HPLC).
is a traditional Indian herb described under the 'Rasayana' class in Ayurveda, which gained immense popularity as a dietary supplement in the USA, Europe, Asia, and the Indian domestic market. ...Despite enormous research on the pharmacological effect of withanosides and withanolides, bioanalytical method development and pharmacokinetics remained challenging and unexplored for these constituents due to isomeric and isobaric characteristics. In current research work, molecular descriptors, pharmacokinetic, and toxicity prediction (ADMET) of these constituents were performed using Molinspiration and admetSAR tools. A rapid, selective, and reproducible bioanalytical method was developed and validated for seven withanosides and withanolides as per USFDA/EMA guidelines, further applied to determine pharmacokinetic parameters of
root extract (WSE) constituents in male
rats at a dose of 500 mg/kg. Additionally, an
permeability study was carried out to explore the absorption pattern of withanosides and withanolides from the intestinal lumen. In silico, ADMET revealed oral bioavailability of withanosides and withanolides following Lipinski's rules of five with significant absorption from the gastrointestinal tract and the ability to cross the blood-brain barrier. Upon oral administration of WSE, C
was found to be 13.833 ± 3.727, 124.415 ± 64.932, 57.536 ± 7.523, and 7.283 ± 3.341 ng/mL for withanoside IV, withaferin A, 12-Deoxy-withastramonolide, and withanolide A, respectively, with T
of 0.750 ± 0.000, 0.250 ± 0.000, 0.291 ± 0.102, and 0.333 ± 0.129 h. Moreover, at a given dose, withanoside V, withanolide B, and withanone were detected in plasma; however, the concentration of these constituents was found below LLOQ. Thus, these four major withanoside and withanolides were quantified in plasma supported by
permeation data exhibiting a time-dependent absorption of withanosides and withanolides across the intestinal barrier. These composite findings provide insights to design a clinical trial of WSE as a potent nutraceutical.
The high-throughput quantification of intact proteoforms using a label-free approach is typically performed on proteins in the 0–30 kDa mass range extracted from whole cell or tissue lysates. ...Unfortunately, even when high-resolution separation of proteoforms is achieved by either high-performance liquid chromatography or capillary electrophoresis, the number of proteoforms that can be identified and quantified is inevitably limited by the inherent sample complexity. Here, we benchmark label-free quantification of proteoforms of Escherichia coli by applying gas-phase fractionation (GPF) via field asymmetric ion mobility spectrometry (FAIMS). Recent advances in Orbitrap instrumentation have enabled the acquisition of high-quality intact and fragmentation mass spectra without the need for averaging time-domain transients prior to Fourier transform. The resulting speed improvements allowed for the application of multiple FAIMS compensation voltages in the same liquid chromatography–tandem mass spectrometry experiment without increasing the overall data acquisition cycle. As a result, the application of FAIMS to label-free quantification based on intact mass spectra substantially increases the number of both identified and quantified proteoforms without penalizing quantification accuracy in comparison to traditional label-free experiments that do not adopt GPF.
The present paper describes a novel two-dimensional liquid chromatography (2D-LC) system, which is comprised of a first-dimensional ion exchange chromatography (IEX1) column, trap column, and ...second-dimensional reversed-phase chromatography (RP2) column system. The biological sample is separated by the first-dimensional LC using an IEX column to remove interferences. The analytes are transferred to the trap column after heart-cutting. Then, the analytes are transferred to the second-dimensional LC using an RP2 column for further separation and ultraviolet detection. This 2D-LC system can offer a large injection volume to provide sufficient sensitivity and exhibits a strong capacity for removing interferences. Here, the determination of three monoterpene indole alkaloids (MIAs; gelsemine, koumine, and humantenmine) from
Gelsemium
in biological matrices (plasma, tissue, and urine) was used this 2D-LC system. After a rapid and easy sample preparation method based on protein precipitation, the sample was injected into the 2D-LC. The method was developed and validated in terms of the selectivity, LOD, LOQ, linearity, precision, accuracy, and stability. The sample preparation time for the three MIAs was 15 min. The LOD for these compounds was 10 ng/mL, which was lower than the developed HPLC methods. The results showed that this method had good quantitation performance and allowed the determination of gelsemine, koumine, and humantenmine in biological matrices. The method is rapid, exhibits high selectivity, has good sensitivity, and is low-cost, thus making it well-suited for application in the pharmaceutical and toxicological analysis of
Gelsemium
.
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