Acid Stress Conditions Research Articles

Stability-indicating UPLC assay coupled with mass spectrometry for the analysis of vilanterol degradation products in human urine

Vilanterol is a once-daily dose inhaler prescribed for asthma and chronic obstructive pulmonary disease. This study involved an investigation of vilanterol stability under acidic, basic, oxidative, thermal, and photolytic stress conditions. UPLC method was developed and validated for the analysis of vilanterol with its degradants. The drug was stable under photolytic and thermal stress conditions and degraded under acidic, basic, and oxidative stress conditions. Degradation kinetics was performed for acidic, basic and oxidative stress conditions. Kinetics parameters, K, half-life time (t1/2) and shelf-life time (t90) were assessed, and the degradation followed first order reaction. The method was linear from 0.10 to 100.00 µg mL−1 with accuracy, inter-day and intra-day precision from 99.45 to 100.02%, 0.391–0.694 and 0.041–0.345, respectively. Mass spectrometry was employed to elucidate the structure of the degradants, and the results revealed that certain degradation products were comparable to vilanterol metabolites. The World Anti-Doping Agency has prohibited the presence of vilanterol and its metabolites in athletes’ urine except for exercise bronchoconstriction with limited dose. So, quantification of vilanterol in the presence of its degradants was performed in human urine. The results revealed that the method was linear in range of 1.00 to 100.00 µg mL−1. Samples collection and experimental protocol was performed according to the guidelines of the Research Ethics Committee of the Faculty of Pharmacy, the British University in Egypt with approval No. CH-2305.

HPLC and LC-MS/MS-Based Quantitative Characterization of Related Substances Associated with Sotalol Hydrochloride.

In total, three related substances (RS) associated with sotalol hydrochloride (STHCl) were herein identified with a novel gradient high-performance liquid chromatography (HPLC) protocol. Further characterization of these substances was then performed via liquid chromatography-mass spectroscopy (LC-MS/MS) and nuclear magnetic resonance (NMR) approaches. For these analyses, commercial STHCl samples were used for quantitative HPLC studies and the degradation of STHCl under acidic (1M HCl), alkaline (1M NaOH), oxidative (30% H2O2), photolytic (4500 Lx), and thermal stress conditions (100 °C) was assessed. This approach revealed this drug to be resistant to acidic, alkaline, and high-temperature conditions, whereas it was susceptible to light and oxidation as confirmed through long-term experiments. The putative mechanisms governing RS formation were also explored, revealing that RS3 was derived from the manufacturing process, whereas RS2 was generated via oxidation and RS1 was generated in response to light exposure. The cytotoxicity of these RS compounds was then assessed using MTT assays and acute toxicity test. Overall, this study provides details regarding the characterization, isolation, quantification, and toxicological evaluation of STHCl and associated RS compounds together with details regarding the precise, specific, and reliable novel HPLC technique, thus providing the requisite information necessary to ensure STHCl purity and safety.

Histidine transport is essential for the growth of Staphylococcus aureus at low pH.

Staphylococcus aureus is an opportunistic pathogen capable of causing many different human diseases. During colonization and infection, S. aureus will encounter a range of hostile environments, including acidic conditions such as those found on the skin and within macrophages. However, little is known about the mechanisms that S. aureus uses to detect and respond to low pH. Here, we employed a transposon sequencing approach to determine on a genome-wide level the genes required or detrimental for growth at low pH. We identified 31 genes that were essential for the growth of S. aureus at pH 4.5 and confirmed the importance of many of them through follow up experiments using mutant strains inactivated for individual genes. Most of the genes identified code for proteins with functions in cell wall assembly and maintenance. These data suggest that the cell wall has a more important role than previously appreciated in promoting bacterial survival when under acid stress. We also identified several novel processes previously not linked to the acid stress response in S. aureus. These include aerobic respiration and histidine transport, the latter by showing that one of the most important genes, SAUSA300_0846, codes for a previously uncharacterized histidine transporter. We further show that under acid stress, the expression of the histidine transporter gene is increased in WT S. aureus. In a S. aureus SAUSA300_0846 mutant strain expression of the histidine biosynthesis genes is induced under acid stress conditions allowing the bacteria to maintain cytosolic histidine levels. This strain is, however, unable to maintain its cytosolic pH to the same extent as a WT strain, revealing an important function specifically for histidine transport in the acid stress response of S. aureus.

Anoxalate decarboxylase-like cupin domain containing protein is involved in imparting acid stress tolerance in Bacillus amyloliquefaciens MBNC.

We report here the structural and functional properties of an oxalate decarboxylase (OxDC)-like cupin domain-containing protein of Bacillus amyloliquefaciens MBNC and its role in imparting tolerance to acid stress conditions. Quantitative real-time PCR (qPCR) analysis revealed 32-fold and 20-fold upregulation of the target gene [(OxDC')cupin] under acetic acid stress and hydrochloric acid stress, respectively, indicating its association with the acid stress response. Bacterial cells with targeted inactivation of the (OxDC')cupin gene using the pMUTIN4 vector system showed decreased growth and survival rate in acidic pH, with drastically reduced exopolysaccharide production. In Silico protein-protein interaction studies revealed seven genes (viz. glmS, nagA, nagB, tuaF, tuaF, gcvT, and ykgA) related to cell wall biosynthesis and biofilm production to interact with OxDC-like cupin domain containing protein. While all these seven genes were upregulated in B. amyloliquefaciens MBNC after 6h of exposure to pH 4.5, the mutant cells containing the inactivated (OxDC')cupingene displayed significantly lower expression (RQ: 0.001-0.02) (compared to the wild-type cells) in both neutral and acidic pH. Our results indicate that the OxDC-like cupin domain containing protein is necessary for cell wall biosynthesis and biofilm production in Bacillus amyloliquefaciens MBNC for survival in acid-stress conditions.

ТOLERANCE TO FREEZE-DRYING CONDITIONS OF Lactobacillus delbrueckii subsp. bulgaricus STRAINS AFTER ACIDIC EXPOSE

The goal of this work was to investigate the effect of fermentation pH and time on the acid tolerance, glycolytic activity, and survival during freeze drying of L. delbrueckii subsp. bulgaricus WDCM 00102 strains during cultivation in the medium, based on pretreated biomass of Spirulina platensis. During the study, when the fermentation process is carried out at a constant pH, a higher value of glycolytic activity is established in the cells of the probiotic culture, which decreases when the pH is lowered. The survival rate of cells of L. delbrueckii subsp. bulgaricus WDCM 00102 after lyophilization shows similar values after cultivation for 25 and 37 h (p < 0.05), while the opposite dependence is established in experiments related to changes in the pH of the cultivation medium. pH and fermentation time significantly influence the glycolytic activity of L. delbrueckii subsp. bulgaricus WDCM 00102 and on the resistance of its cells to acid stress and lyophilization conditions.

STABILITY INDICATING UPLC METHOD FOR ESTIMATION OF BENAZEPRIL AND HYDROCHLOROTHIAZIDE IN BULK AND COMBINED DOSAGE FORM

Objective: The main objective was to develop stability indicating UPLC technique for simultaneous estimation of Benazepril and Hydrochlorothiazide in bulk and formulation. Methods: 0.1% Triethylamine phosphate: Methanol (25:75v/v) was used as the mobile phase. Benazepril linearity was found to be 4-20 µg/ml and Hydrochlorothiazide linearity was found to be 5-25 g/ml. The detection wavelength was 236 nm, and the retention period of Benazepril was 3.4 min and Hydrochlorothiazide was 5.4 min with a flow rate of 1.0 ml/min. According to the ICH guidlines, the proposed method was validated and stress studies revealed that the drugs are prone to alkali and peroxide stress conditions. Results: The calibration curve was plotted, and the regression equations for Benazepril were y = 2,01,491.67x+60,532.30 with a correlation coefficient (r2) of 0.9997 and Hydrochlorothiazide were y = 64,635.86x-74,607.10 with a correlation coefficient (r2) of 0.9994. According to the accuracy research, the percent recovery of Benazepril is 99.09-100.69 % and that of Hydrochlorothiazide is 98.27-101.88%, both of which are within the ICH recommendations. Benazepril has a limit of detection of 0.08 g/ml-0.24 g/ml and Hydrochlorothiazide has a limit of quantitation of 0.03 g/ml-0.10 g/ml. The procedure was found to be straightforward, linear, rapid, exact, repeatable, and robust. It was determined that the % RSD was within ICH norms. Stress degradation tests showed the drug's vulnerability to oxidative, thermal, photolytic, acid, basic, and neutral hydrolysis stress conditions. Under the circumstances of alkali and peroxide stress, it was discovered that the drug degraded most quickly. Conclusion: The developed chromatographic technique under consideration was suitable for the accurate, precise, and quick simultaneous measurement of hydrochlorothiazide and benazepril in both their bulk and medicinal dose forms.

The RNA chaperone Hfq has a multifaceted role in Edwardsiella ictaluri.

Edwardsiella ictaluri is a Gram-negative, facultative intracellular bacterium that causes enteric septicemia in catfish (ESC). The RNA chaperone Hfq (host factor for phage Qβ replication) facilitates gene regulation via small RNAs (sRNAs) in various pathogenic bacteria. Despite its significance in other bacterial species, the role of hfq in E. ictaluri remains unexplored. This study aimed to elucidate the role of hfq in E.ictaluri by creating an hfq mutant (EiΔhfq) through in-frame gene deletion and characterization. Our findings revealed that the Hfq protein is highly conserved within the genus Edwardsiella. The deletion of hfq resulted in a significantly reduced growth rate during the late exponential phase. Additionally, EiΔhfq displayed a diminished capacity for biofilm formation and exhibited increased motility. Under acidic and oxidative stress conditions, EiΔhfq demonstrated impaired growth, and we observed elevated hfq expression when subjected to in vitro and in vivo stress conditions. EiΔhfq exhibited reduced survival within catfish peritoneal macrophages, although it had no discernible effect on the adherence and invasion of epithelial cells. The infection model revealed that hfq is needed for bacterial persistence in catfish, and its absence caused significant virulence attenuation in catfish. Finally, the EiΔhfq vaccination completely protected catfish against subsequent EiWT infection. In summary, these results underscore the pivotal role of hfq in E. ictaluri, affecting its growth, motility, biofilm formation, stress response, and virulence in macrophages and within catfish host.

Stability-Indicating Liquid Chromatography Method for Rifaximin and LC-MS Characterization of its Potential Degradants

Rifaximin (RFX) is a structural analog of rifampin that has been shown to be a gastrointestinal-selective antibiotic with broad-spectrum antibacterial properties, a pronounced safety profile, and limited drug interactions. Crohn’s disease, diarrhea, hepatic encephalopathy, irritable bowel syndrome, and traveler’s diarrhea caused by non-invasive diarrheagenic Escherichia coli, can all be treated with rifaximin. FDA has designated RFX as an orphan drug for the treatment of hepatic encephalopathy. There is a substantial need for analytical quality monitoring of Rifaximin to ensure the safety and efficacy of treatment. To generate degradants, forced degradation study of the drug was carried out in acid, alkali, oxidative, thermal, and photolytic stress conditions. The optimum separation of the drug, and its degradants was achieved on an Inertsil ODS-3V C18 column (250 X 4.6 mm X 5 μm) under gradient elution conditions using, 10 mM potassium dihydrogen orthophosphate (pH 5.0 ± 0.05) as mobile phase A, and acetonitrile as mobile phase B, at a flow rate of 1.0 mL/min. The run time was 41 minutes and the detection wavelength was 258 nm. The method was validated in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human use Guidelines. The drug was stable in neutral hydrolysis, photo and dry heat degradation conditions. The percentage degradation observed during acid, alkali hydrolysis and oxidative stress conditions were 70.46, 15.11 and 24.18, respectively. The degradants were investigated by LC-MS which indicated the presence of four (m/z 784.2, 744.5, 784.3, 753.8), three (m/z 744.5, 784.3, 753.8) and five (m/z 772.4, 838.4, 744.5, 753.8, and 801.9) degradants in acid, alkali and oxidative stress conditions, respectively. The structures of degradants were elucidated using Liquid chromatography–mass spectrometry (LC-MS) and Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses.

Prebiotic Cellulose-Pullulan Matrix as a "Vehicle" for Probiotic Biofilm Delivery to the Host Large Intestine.

This study describes the development of a new combined polysaccharide-matrix-based technology for the immobilization of Lactobacillus rhamnosus GG (LGG) bacteria in biofilm form. The new composition allows for delivering the bacteria to the digestive tract in a manner that improves their robustness compared with planktonic cells and released biofilm cells. Granules consisting of a polysaccharide matrix with probiotic biofilms (PMPB) with high cell density (>9 log CFU/g) were obtained by immobilization in the optimized nutrient medium. Successful probiotic loading was confirmed by fluorescence microscopy and scanning electron microscopy. The developed prebiotic polysaccharide matrix significantly enhanced LGG viability under acidic (pH 2.0) and bile salt (0.3%) stress conditions. Enzymatic extract of feces, mimicking colon fluid in terms of cellulase activity, was used to evaluate the intestinal release of probiotics. PMPB granules showed the ability to gradually release a large number of viable LGG cells in the model colon fluid. In vivo, the oral administration of PMPB granules in rats resulted in the successful release of probiotics in the colon environment. The biofilm-forming incubation method of immobilization on a complex polysaccharide matrix tested in this study has shown high efficacy and promising potential for the development of innovative biotechnologies.

Identification of degradation products of brivaracetam using liquid chromatography quadrupole time-of-flight tandem mass spectrometry: Degradation pathway elucidation.

Pyrrolidone-based drugs find widespread use in treating conditions such as epilepsy and Alzheimer's disease, and in various other medical applications. Brivaracetam, the latest generation of pyrrolidone drugs, has exhibited significant promise owing to chemical structure modifications. Its affinity to the SV2A receptor is double that of the previous-generation drug, levetiracetam. Consequently, brivaracetam holds substantial potential for diverse applications. As a novel drug not yet included in the pharmacopeias of developed nations, comprehensive analysis and research are necessary to guarantee its safe utilization in clinical settings. A liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC/QTOFMS) method has been developed to effectively separate, identify and characterize both the degradation products and process-related substances of brivaracetam. Stress testing of the sample was carried out following the guidelines outlined in ICH Q1A(R2). The structures of these impurities were identified through positive electrospray ionization QTOF high-resolution MS and NMR spectroscopy. Additionally, the formation mechanism of each degradation product is thoroughly discussed. Under the analytical conditions outlined in this paper, brivaracetam and its degradation products were effectively separated. Thirteen degradation products were detected and characterized, shedding light on their origins and degradation pathways. Among these, three degradation products align with previously reported impurities, and two unreported degradation products were synthesized and confirmed through NMR spectroscopy. The stress testing results revealed the instability of brivaracetam under acidic, alkaline, oxidative and thermal stress conditions, while it exhibited relative stability under photolytic stress conditions. The study developed an analytical method for brivaracetam that enabled the effective detection and separation of brivaracetam and its 13 degradation products. This method addresses a gap in both current domestic and foreign drug standards. The structures of all the major degradation products were characterized by high-resolution LC/QTOFMS, which is essential for quality control during the drug production process, stability evaluation and the establishment of proper storage conditions.

Ribosome profiling reveals the fine-tuned response of Escherichia coli to mild and severe acid stress.

Bacteria react very differently to survive in acidic environments, such as the human gastrointestinal tract. Escherichia coli is one of the extremely acid-resistant bacteria and has a variety of acid-defense mechanisms. Here, we provide the first genome-wide overview of the adaptations of E. coli K-12 to mild and severe acid stress at both the transcriptional and translational levels. Using ribosome profiling and RNA sequencing, we uncover novel adaptations to different degrees of acidity, including previously hidden stress-induced small proteins and novel key transcription factors for acid defense, and report mRNAs with pH-dependent differential translation efficiency. In addition, we distinguish between acid-specific adaptations and general stress response mechanisms using denoising autoencoders. This workflow represents a powerful approach that takes advantage of next-generation sequencing techniques and machine learning to systematically analyze bacterial stress responses.

Eco-Friendly Stability Indicating HPLC Method for Estimation of Elbasvir and Grazoprevir by Quality by Design Approach

Introduction: The pharmaceutical industry faces a significant problem as a result of the worldwide requirement to modify processes in order to comply with the green analytical chemistry (GAC) requirements. An enormous amount of organic hazardous waste is produced by high-performance liquid chromatography (HPLC), one of the methods employed the most frequently at different stages in the pharmaceutical sector. Aim: To develop analytical quality by design-aided stability indicating green high-performance liquid chromatography (HPLC) method for estimation of Elbasvir and Grazoprevir in a dosage form. Material and Methods: The critical chromatographic factors were the % of ethanol in the mobile phase, flow rate, and their overall effect on the responses like asymmetry, theoretical plates, and resolution were studied to optimize the method. Green analytical chemistry (GAC) has mainly focused on developing analytical methods that are safe for the environment. Therefore, it is imperative that the GAC principles be applied in pharmacological analysis. A rotatable central composite design was employed, and the optimized conditions for chromatographic separation were made with a run time of 5 minutes using Zorbax C18 column (4.6× 150 mm, 5 µm) with 0.1% Trifluoroacetic acid and ethanol (40:60 v/v) as components of a mobile phase, flowing at a rate of 1.0 ml/minute. Photodiode array detection was carried out at 253 nm. Results: The retention time was 1.8 min for EBS and 2.84 min for GZP. According to the ICH guidelines, the proposed method was validated and stress studies revealed that Elbasvir and Grazoprevir are prone to acidic, basic, and oxidation stress conditions. An analytical eco-scale score evaluated the greenness profile and a software-based evaluation. Conclusion: The developed HPLC method is eco-friendly and shall be adopted in the routine quality control of Elbasvir and Grazoprevir in a tablet dosage form.

Transcriptomics of Lactobacillus paracasei: metabolism patterns and cellular responses under high-density culture conditions.

Lactobacillus paracasei has significant potential for development and application in the environmental field, particularly in addressing malodor pollution. This study aims to investigate the cellular response of L. paracasei B1 under high-density culture conditions. The selected strain has previously shown effective deodorizing and bacteriostatic abilities. Transcriptomics techniques are employed to dissect the nutrient metabolism pattern of L. paracasei B1 and its response mechanism under environmental stress. The study characterizes the functions of key differentially expressed genes during growth before and after optimizing the culture conditions. The optimization of fermentation culture conditions provides a suitable growth environment for L. paracasei B1, inducing an enhancement of its phosphotransferase system for sugar source uptake and maintaining high levels of glycolysis and pyruvate metabolism. Consequently, the strain is able to grow and multiply rapidly. Under acid stress conditions, glycolysis and pyruvate metabolism are inhibited, and L. paracasei B1 generates additional energy through aerobic respiration to meet the energy demand. The two-component system and quorum sensing play roles in the response and regulation of L. paracasei B1 to adverse environments. The strain mitigates oxygen stress damage through glutathione metabolism, cysteine and methionine metabolism, base excision repair, and purine and pyrimidine metabolism. Additionally, the strain enhances lysine synthesis, the alanine, aspartate, and glutamate metabolic pathways, and relies on the ABC transport system to accumulate amino acid-compatible solutes to counteract acid stress and osmotic stress during pH regulation. These findings establish a theoretical basis for the further development and application of L. paracasei B1 for its productive properties.

Eco-Friendly High-Performance Thin-Layer Chromatography Method for the Determination of Tenoxicam in Commercial Formulations.

There is a dearth of information in the literature regarding environmentally benign high-performance thin-layer chromatography (HPTLC) methods to determine tenoxicam (TNX). Therefore, designing and validating an HPTLC method to detect TNX in commercial tablets and capsules was the goal of this investigation. The green mobile phase utilized was the combination of ethanol/water/ammonia solution (50:45:5 v/v/v). The TNX was quantified at a wavelength of 375 nm. The proposed method's greenness profile was established using the Analytical GREEnness (AGREE) approach. The proposed methodology for determining TNX was linear in the range of 25-1400 ng/band. The proposed methodology for measuring TNX was accurate (% recoveries = 98.24-101.48), precise (% RSD = 0.87-1.02), robust (% RSD = 0.87-0.94), sensitive (LOD = 0.98 ng/band and LOQ = 2.94 ng/band), and environmentally friendly. The AGREE scale for the present methodology was derived to be 0.75, indicating an outstanding greenness profile. TNX was found to be highly stable under acidic, base, and thermal stress conditions. However, it completely decomposed under oxidative stress conditions. Commercial tablets and capsules were found to have 98.46 and 101.24% TNX, respectively. This finding supports the validity of the current methodology for measuring TNX in commercial formulations. The outcomes of this work showed that the proposed eco-friendly HPTLC methodology can be used for the routine analysis of TNX in commercial formulations.

A comprehensive investigation of protein expression profiles in L. monocytogenes exposed to thermal abuse, mild acid, and salt stress conditions.

Preventing L. monocytogenes infection is crucial for food safety, considering its widespread presence in the environment and its association with contaminated RTE foods. The pathogen's ability to persist under adverse conditions, for example, in food processing facilities, is linked to virulence and resistance mechanisms, including biofilm formation. In this study, the protein expression patterns of two L. monocytogenes 1/2a strains, grown under environmental stressors (mild acidic pH, thermal abuse, and high concentration of NaCl), were investigated. Protein identification and prediction were performed by nLC-ESI-MS/MS and nine different bioinformatic software programs, respectively. Gene enrichment analysis was carried out by STRING v11.05. A total of 1,215 proteins were identified, of which 335 were non-cytosolic proteins and 265 were immunogenic proteins. Proteomic analysis revealed differences in protein expression between L. monocytogenes strains in stressful conditions. The two strains exhibited unique protein expression profiles linked to stress response, virulence, and pathogenesis. Studying the proteomic profiles of such microorganisms provides information about adaptation and potential treatments, highlighting their genetic diversity and demonstrating the utility of bioinformatics and proteomics for a broader analysis of pathogens.

The Transcription Factor CsgD Contributes to Engineered Escherichia coli Resistance by Regulating Biofilm Formation and Stress Responses.

The high cell density, immobilization and stability of biofilms are ideal characteristics for bacteria in resisting antibiotic therapy. CsgD is a transcription activating factor that regulates the synthesis of curly fimbriae and cellulose in Escherichia coli, thereby enhancing bacterial adhesion and promoting biofilm formation. To investigate the role of CsgD in biofilm formation and stress resistance in bacteria, the csgD deletion mutant ΔcsgD was successfully constructed from the engineered strain E. coli BL21(DE3) using the CRISPR/Cas9 gene-editing system. The results demonstrated that the biofilm of ΔcsgD decreased by 70.07% (p < 0.05). Additionally, the mobility and adhesion of ΔcsgD were inhibited due to the decrease in curly fimbriae and extracellular polymeric substances. Furthermore, ΔcsgD exhibited a significantly decreased resistance to acid, alkali and osmotic stress conditions (p < 0.05). RNA-Seq results revealed 491 differentially expressed genes between the parent strain and ΔcsgD, with enrichment primarily observed in metabolism-related processes as well as cell membrane structure and catalytic activity categories. Moreover, CsgD influenced the expression of biofilm and stress response genes pgaA, motB, fimA, fimC, iraP, ompA, osmC, sufE and elaB, indicating that the CsgD participated in the resistance of E. coli by regulating the expression of biofilm and stress response. In brief, the transcription factor CsgD plays a key role in the stress resistance of E. coli, and is a potential target for treating and controlling biofilm.

A new stability-indicating HPLC-UV method for determination of amlodipine besylate and its impurities in drug substance

A new fast stability-indication high performance liquid chromatography method was developed and validated for the determination of amlodipine besylate and its organic impurities in drug substance. The separation of amlodipine and its seven impurities was achieved on a core shell C18 column, 100 mm × 4.6 mm; 2.6 μm, within 15 min. The mobile phase comprised of 0.4% ammonium hydroxide in water and methanol delivered in a gradient mode; the method detection wavelength is 237 nm. The selected column is stable at high pH and provided a good peak shape for basic compounds. Amlodipine besylate was subject to acid, base, oxidative, thermal, and photolytic stress conditions. The degradation products were well resolved from the amlodipine peak and its impurities. Major degradants were analyzed by liquid chromatography coupled with single-quadrupole mass detector. Amlodipine peak was shown to be free of co-elution by mass spectral analysis in all stress conditions. The method was validated in terms of specificity, linearity, accuracy, precision, and robustness. The developed method could be applied for routine quality control analysis of amlodipine besylate drug substance.

Hesperidin: Enrichment, forced degradation, and structural elucidation of potential degradation products using spectral techniques.

Hesperidin (HES) is a well-known citrus bioflavonoid phyto-nutraceutical agent with polypharmacological properties. After 2019, HES was widely used for prophylaxis and COVID-19 treatment. Moreover, it is commonly prescribed for treating varicose veins and other diseases in routine clinical practice. Pharmaceutical impurities and degradation products (DP) impact the drug's quality and safety and thus its effectiveness. Therefore, forced degradation studies help study drug stability, degradation mechanisms, and their DPs. This study was performed because stress stability studies using detailed structural characterization of hesperidin are currently unavailable in the literature. In the HES enrichment method crude HES was converted to its pure form (98% purity) using column chromatography and then subjected to forced degradation under acid, base, and neutral hydrolyses followed by oxidative, reductive, photolytic, and thermal stress testing (International Conference on Harmonization guidelines). The stability-indicating analytical method (SIAM) was developed to determine DPs using reversed-phase high-performance liquid chromatography (C18 column with methanol and 0.1% v/v acetic acid in deionized water [70:30, v/v] at 284 nm). Further, structural characterization of DPs was performed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. In addition, in silico toxicity predictions were performed using pKCSM and DataWarior freeware. HES was found to be susceptible to acidic and basic hydrolytic conditions and yielded three DPs in each, which were detected using designed SIAM. Of six DPs, three were pseudo-DPs (short lived), and the remaining were characterized using LC-MS/MS and NMR spectroscopy. The tentative mechanism of the formation of proposed DPs was explained. The proposed DPs were found inactive from in silico toxicity predictions. Hesperidin was labile under acidic and basic stress conditions. The potential DPs were characterized using LC-ESI-MS/MS and NMR spectral techniques. The proposed mechanism of formation was hypothesized. In addition, to identify and characterize the DPs, a SIAM, which has broad biomedical applications, was successfully developed.

Membrane lipid and expression responses of Saccharolobus islandicus REY15A to acid and cold stress.

Archaea adjust the number of cyclopentane rings in their glycerol dibiphytanyl glycerol tetraether (GDGT) membrane lipids as a homeostatic response to environmental stressors such as temperature, pH, and energy availability shifts. However, archaeal expression patterns that correspond with changes in GDGT composition are less understood. Here we characterize the acid and cold stress responses of the thermoacidophilic crenarchaeon Saccharolobus islandicus REY15A using growth rates, core GDGT lipid profiles, transcriptomics and proteomics. We show that both stressors result in impaired growth, lower average GDGT cyclization, and differences in gene and protein expression. Transcription data revealed differential expression of the GDGT ring synthase grsB in response to both acid stress and cold stress. Although the GDGT ring synthase encoded by grsB forms highly cyclized GDGTs with ≥5 ring moieties, S. islandicus grsB upregulation under acidic pH conditions did not correspond with increased abundances of highly cyclized GDGTs. Our observations highlight the inability to predict GDGT changes from transcription data alone. Broader analysis of transcriptomic data revealed that S. islandicus differentially expresses many of the same transcripts in response to both acid and cold stress. These included upregulation of several biosynthetic pathways and downregulation of oxidative phosphorylation and motility. Transcript responses specific to either of the two stressors tested here included upregulation of genes related to proton pumping and molecular turnover in acid stress conditions and upregulation of transposases in cold stress conditions. Overall, our study provides a comprehensive understanding of the GDGT modifications and differential expression characteristic of the acid stress and cold stress responses in S. islandicus.

The occurrence and resistance of Shigella flexneri CECT4804 to acid stress in vitro and in vivo.

The ability to maintain intra‑cellular pH is crucial for many microbes mainly the enterobacteria to survive in diverse environments, particularly those that undergo fluctuations in pH. In this context, the growth and survival of Shigella flexneri at different acid pH values were evaluated to explain the response strategies involved in the adaptation of S. flexneri CECT4804 in acid stress conditions. Furthermore, the capacity of this strain to produce slime on Congo Red Agar, biofilm formation on polystyrene plate and hydrophobicity are reported. In addition, the modification of the membrane fatty acids profiles has been studied. Moreover, an infection with the stressed strain was realized on rats in rates and examined for their toxicity in intestine tissue. The obtained results show that the strain survival is strongly influenced by acidity. Indeed, the stressed and unstressed strains became biofilm positive after acid stress. A significant increase in the hydrophobicity percentage compared to the values found under normal conditions, is also noticed. For the membrane fatty acids analysis, the acidity induces several modifications in the membrane composition. After the infection, the gravest lesion was registered in the intestine of rats administered with the bacteria stressed at the lowest pH.