Changes in Metabolite Profiles of Chinese Soy Sauce at Different Time Durations of Fermentation Studied by 1H-NMR-Based Metabolomics.

Fermentation parameters, especially the duration, are important in imparting a peculiar taste and flavor to soy sauce. The main purpose of this research was to monitor metabolic changes occurring during the various time intervals of the fermentation process. NMR-based metabolomics was used to monitor the compositional changes in soy sauce during fermentation. The 1H-NMR spectra of the soy sauce samples taken from the fermentation tanks at 0 to 8 months were analyzed using 1H-NMR spectroscopy, and the obtained spectra were analyzed by multivariate statistical analysis. The Principal Component Analysis (PCA) and Partial Least Square Discriminate analysis (PLSDA) revealed the separation of samples fermented for various time durations under identical conditions. Key metabolites shown by corresponding loading plots exhibited variations in amino acids (lysine, threonine, isoleucine, etc.), acetate, glucose, fructose, sucrose, ethanol, glycerol, and others. The levels of ethanol in soy sauce increased with longer fermentation durations, which can be influenced by both natural fermentation and the intentional addition of ethanol as a preservative. The study shows that the variation in metabolite can be very efficiently monitored using 1H-NMR-based metabolomics, thus suggestion to optimize the time duration to get the soy sauce product with the desired taste and flavor.

Investigation of Pharmacologically Important Polyphenolic Secondary Metabolites in Plant-based Food Samples Using HPLC-DAD.

Polyphenolic compounds are vital components of plants. However, their analysis is particularly difficult and challenging due to their similar chemical and structural properties. In this study, we developed a simple and reproducible HPLC-DAD protocol for determining nineteen pharmacologically important polyphenols in plant-based food samples, including fruits (apple, banana, grapefruit, peach, grapes, plum, and pear), vegetables (onion, cabbage, capsicum, garlic, lemon, tomato, potato, and spinach), and other edible items (corn, kidney beans, green tea, black tea, and turmeric). The reference standards were pooled into four different groups based on logP values and expected retention time to avoid compound co-elution. These developed methods will be useful for the qualitative and quantitative analysis of biologically important polyphenolic compounds in various food samples and botanicals.

Studying molecular interactions via capillary electrophoresis and microscale thermophoresis: A review.

The process of choosing the most proper technique for studying the molecular interactions is based on critical factors such as instrumentation complexity, automation, experimental procedures, analysis time, consumables, and cost-value. This review has tracked the use of affinity capillary electrophoresis (ACE) and microscale thermophoresis (MST) techniques in the evaluation of molecular binding among different molecules during the 5 years 2016-2021. ACE has proved to be an attractive technique for biomolecular characterization with high resolution efficiency where small variations in several controlling factors can much improve such efficiency compared to other analytical techniques. Meanwhile, MST has proved its higher sensitivity for smaller amounts of complex non-purified biosamples without affecting its robustness while providing high through output. However, the main motivation to review both techniques in the proposed review was their capability to carry out all experiments without the need for immobilizing one interacting partner, besides a great flexibility in the use of buffering systems. The proposed review demonstrates the importance of both techniques in different areas of life sciences. Moreover, the recent advances in exploiting ACE and MST in other research interests have been discussed.

Computational 3D Modeling-Based Identification of Inhibitors Targeting Cysteine Covalent Bond Catalysts for JAK3 and CYP3A4 Enzymes in the Treatment of Rheumatoid Arthritis.

This work aimed to find new inhibitors of the CYP3A4 and JAK3 enzymes, which are significant players in autoimmune diseases such as rheumatoid arthritis. Advanced computer-aided drug design techniques, such as pharmacophore and 3D-QSAR modeling, were used. Two strong 3D-QSAR models were created, and their predictive power was validated by the strong correlation (R2 values > 80%) between the predicted and experimental activity. With an ROC value of 0.9, a pharmacophore model grounded in the DHRRR hypothesis likewise demonstrated strong predictive ability. Eight possible inhibitors were found, and six new inhibitors were designed in silico using these computational models. The pharmacokinetic and safety characteristics of these candidates were thoroughly assessed. The possible interactions between the inhibitors and the target enzymes were made clear via molecular docking. Furthermore, MM/GBSA computations and molecular dynamics simulations offered insightful information about the stability of the binding between inhibitors and CYP3A4 or JAK3. Through the integration of various computational approaches, this study successfully identified potential inhibitor candidates for additional investigation and efficiently screened compounds. The findings contribute to our knowledge of enzyme-inhibitor interactions and may help us create more effective treatments for autoimmune conditions like rheumatoid arthritis.

Biochemical and in silico inhibition of bovine and human carbonic anhydrase-II by 1H-1,2,3-triazole analogs.

A series of 1H-1,2,3-triazole analogs (7a-7d and 9a-9s) were synthesized via "click" chemistry and evaluated for in vitro carbonic anhydrase-II (bovine and human) inhibitory activity. The synthesis of intermediates, 7a and 7c, was achieved by using (S)-(-)ethyl lactate as a starting material. These compounds (7a and 7c) underwent Suzuki-Miyaura cross-coupling reaction with different arylboronic acids in 1,4-dioxane, reflux at 90-120°C for 8 h using Pd(PPh3)4 as a catalyst (5 mol%), and K2CO3 (3.0 equiv)/K2PO4 (3.0 equiv) as a base to produce target 1H-1,2,3-triazole molecules (9a-9s) for a good yield of 67-86%. All the synthesized compounds were characterized through NMR spectroscopic techniques. Furthermore, all those compounds have shown significant inhibitory potential for both sources of carbonic anhydrase-II (CA-II). In the case of bCA-II, compounds 9i, 7d, 9h, 9o, 9g, and 9e showed potent activity with IC50 values in the range of 11.1-17.8 µM. Whereas for hCA-II, compounds 9i, 9c, 9o, and 9j showed great potential with IC50 values in the range of 10.9-18.5 µM. The preliminary structure-activity relationship indicates that the presence of the 1H-1,2,3-triazole moiety in those synthesized 1H-1,2,3-triazole analogs (7a-7d and 9a-9s) significantly contributes to the overall activity. However, several substitutions on this scaffold affect the activity to several folds. The selectivity index showed that compounds 9c, 9k, and 9p are selective inhibitors of hCA-II. Kinetics studies showed that these compounds inhibited both enzymes (bCA-II and hCA-II) in a competitive manner. Molecular docking indicates that all the active compounds fit well in the active site of CA-II. This study has explored the role of 1H-1,2,3-triazole-containing compounds in the inhibition of CA-II to combat CA-II-related disorders.

Metabolomics Study of Serum Samples of ß-YAC Transgenic Mice Treated with Tenofovir Disoproxil Fumarate.

ß-thalassemia is one of the most common monogenic disorders and a life-threatening health issue in children. A cost-effective and safe therapeutic approach to treat this disease is to reactivate the γ-globin gene for fetal hemoglobin (HbF) production that has been silenced during infancy. Hydroxyurea (HU) is the only FDA approved HbF inducer. However, its cytotoxicity and inability to respond significantly in all patients pose a need for an HbF inducer with better efficacy. The study describes the serum metabolic alteration in ß-YAC transgenic mice treated with Tenofovir disoproxil fumarate (TDF) (n = 5), a newly identified HbF inducer, and compared to the mice groups treated with HU (n = 5) and untreated control (n = 5) using gas chromatography-mass spectrometry. Various univariate and multivariate statistical analyses were performed to identify discriminant metabolites that altered the biological pathways encompassing galactose metabolism, lactose degradation, and inositol. Furthermore, the decreased concentrations of L-fucose and geraniol in TDF-treated mice help in recovering towards normal, decreasing oxidative stress even much better than the HU-treated mice. The proposed study suggested that TDF can reduce the deficiency of blood required for ß-thalassemia and can be used for the preclinical study at phase I/II for fetal hemoglobin production.

Microscale Thermophoresis and Molecular Modelling to Explore the Chelating Drug Transportation in the Milk to Infant.

The microscale thermophoresis (MST) technique was utilized to investigate lactoferrin-drug interaction with the iron chelator, deferiprone, using label-free system. MST depends on the intrinsic fluorescence of one interacting partner. The results indicated a significant interaction between lactoferrin and deferiprone. The estimated binding constant for the lactoferrin-deferiprone interaction was 8.9 × 10-6 ± 1.6, SD, which is to be reported for the first time. Such significant binding between lactoferrin and deferiprone may indicate the potentiation of the drug secretion into a lactating mother's milk. The technique showed a fast and simple approach to study protein-drug interaction while avoiding complicated labeling procedures. Moreover, the binding behavior of deferiprone within the binding sites of lactoferrin was investigated through molecular docking which reflected that deferiprone mediates strong hydrogen bonding with ARG121 and ASP297 in pocket 1 and forms H-bond and ionic interaction with ASN640 and ASP395, respectively, in pocket 2 of lactoferrin. Meanwhile, iron ions provide ionic interaction with deferiprone in both of the pockets. The molecular dynamic simulation further confirmed that the binding of deferiprone with lactoferrin brings conformational changes in lactoferrin that is more energetically stable. It also confirmed that deferiprone causes positive correlation motion in the interacting residues of both pockets, with strong negative correlation motion in the loop regions, and thus changes the dynamics of lactoferrin. The MM-GBSA based binding free energy calculation revealed that deferiprone exhibits ∆G TOTAL of -63,163 kcal/mol in pocket 1 and -63,073 kcal/mol in pocket 2 with complex receptor-ligand difference in pocket 1 and pocket 2 of -117.38 kcal/mol and -111.54 kcal/mol, respectively, which in turn suggests that deferiprone binds more strongly in the pocket 1. The free energy landscape of the lactoferrin-deferiprone complex also showed that this complex remains in a high energy state that confirms the strong binding of deferiprone with the lactoferrin. The current research concluded that iron-chelating drugs (deferiprone) can be transported from the mother to the infant in the milk because of the strong attachment with the lactoferrin active pockets.

Sensitive Detection of Pharmaceutical Drugs and Metabolites in Serum Using Data-Independent Acquisition Mass Spectrometry and Open-Access Data Acquisition Tools.

Data-independent acquisition (DIA) based strategies have been explored in recent years for improving quantitative analysis of metabolites. However, the data analysis is challenging for DIA methods as the resulting spectra are highly multiplexed. Thus, the DIA mode requires advanced software analysis to facilitate the data deconvolution process. We proposed a pipeline for quantitative profiling of pharmaceutical drugs and serum metabolites in DIA mode after comparing the results obtained from full-scan, Data-dependent acquisition (DDA) and DIA modes. using open-access software. Pharmaceutical drugs (10) were pooled in healthy human serum and analysed by LC-ESI-QTOF-MS. MS1 full-scan and Data-dependent (MS2) results were used for identification using MS-DIAL software while deconvolution of MS1/MS2 spectra in DIA mode was achieved by using Skyline software. The results of acquisition methods for quantitative analysis validated the remarkable analytical performance of the constructed workflow, proving it to be a sensitive and reproducible pipeline for biological complex fluids.

Formulation Development and In Vitro/In Vivo Characterization of Methotrexate-Loaded Nanoemulsion Gel Formulations for Enhanced Topical Delivery.

Methotrexate-loaded oil-in-water nanoemulsion formulations were prepared using the high shear homogenization technique. A drug excipient study (ATR-FTIR) was carried out to investigate the compatibility between the drug, the polymers, and its admixtures. The thermal stability of the nanoemulsion formulations was evaluated by subjecting them to a heating and cooling cycle. The prepared nanoemulsion formulations (FNE1 to FNE6) were evaluated for particle size, PDI value, and entrapment efficiency (EE). They were analyzed for morphological information using transmission electron microscopy. The drug (methotrexate)-loaded nanoemulsion formulations (FNE2, FNE4, and FNE6) were then converted into nanoemulsion gel formulations by adding 1% chitosan (polymer) as a gelling agent. The nanoemulsion gel formulations (FNEG2, FNEG4, and FNEG6) were investigated for physicochemical parameters, viscosity, spreadability, extrudability, drug content, and skin irritation. Various penetration enhancers (olive oil, clove, and almond oil) were employed to examine the potency of the prepared nanoemulsion gel formulations. In vitro drug release, ex vivo permeation, skin drug retention, and stability tests were carried out for evaluation of the prepared nanoemulsion gel formulations (FNEG2, FNEG4, and FNEG6). The data obtained from the in vitro study were subjected to the kinetic model, and the Korsemeyer-Peppas model was best fitted to the data. The nanoemulsion gel formulation FNEG6 showed the maximum controlled drug release and followed an anomalous, non-Fickian release mechanism. The use of almond oil in the preparation of the nanoemulsion gel formulation FNEG6 helped the penetration of the drug across stratum corneum and the restructuring of the properties of skin and resulted in a higher penetration and retention of methotrexate in a deeper layer of the skin. The current study concluded that the methotrexate-loaded nanoemulsion gel formulation FNEG6 showed the best optimum release, permeation, and retention results as compared to the available oral tablets' formulations, followed by a low serum concentration and the maximum drug retention, which is beneficial in treating skin infections and reducing systemic toxicity.

Chiral Monolithic Silica-Based HPLC Columns for Enantiomeric Separation and Determination: Functionalization of Chiral Selector and Recognition of Selector-Selectand Interaction.

This review draws attention to the use of chiral monolithic silica HPLC columns for the enantiomeric separation and determination of chiral compounds. Properties and advantages of monolithic silica HPLC columns are also highlighted in comparison to conventional particle-packed, fused-core, and sub-2-µm HPLC columns. Nano-LC capillary monolithic silica columns as well as polymeric-based and hybrid-based monolithic columns are also demonstrated to show good enantioresolution abilities. Methods for introducing the chiral selector into the monolithic silica column in the form of mobile phase additive, by encapsulation and surface coating, or by covalent functionalization are described. The application of molecular modeling methods to elucidate the selector-selectand interaction is discussed. An application for enantiomeric impurity determination is also considered.

Immobilization of Chondroitin Sulfate A onto Monolithic Epoxy Silica Column as a New Chiral Stationary Phase for High-Performance Liquid Chromatographic Enantioseparation.

Chondroitin sulfate A was covalently immobilized onto a monolithic silica epoxy column involving a Schiff base formation in the presence of ethylenediamine as a spacer and evaluated in terms of its selectivity in enantioseparation. The obtained column was utilized as a chiral stationary phase in enantioseparation of amlodipine and verapamil using a mobile phase consisting of 50 mM phosphate buffer pH 3.5 and UV detection. Sample dilution by organic solvents (preferably 25% v/v acetonitrile-aqueous solution) was applied to achieve baseline enantioresolution (Rs > 3.0) of the individual drug models within 7 min, an excellent linearity (R2 = 0.999) and an interday repeatability of 1.1% to 1.8% RSD. The performance of the immobilized column for quantification of racemate in commercial tablets showed a recovery of 86-98% from tablet matrices. Computational modeling by molecular docking was employed to investigate the feasible complexes between enantiomers and the chiral selector.

Thermophoresis for characterizing biomolecular interaction.

The study of biomolecular interactions is crucial to get more insight into the biological system. The interactions of protein-protein, protein-nucleic acids, protein-sugars, nucleic acid-nucleic acids and protein-small molecules are supporting therapeutics and technological developments. Recently, the development in a large number of analytical techniques for characterizing biomolecular interactions reflect the promising research investments in this field. In this review, microscale thermophoresis technology (MST) is presented as an analytical technique for characterizing biomolecular interactions. Recent years have seen much progress and several applications established. MST is a powerful technique in quantitation of binding events based on the movement of molecules in microscopic temperature gradient. Simplicity, free solutions analysis, low sample volume, short analysis time, and immobilization free are the MST advantages over other competitive techniques. A wide range of studies in biomolecular interactions have been successfully carried out using MST, which tend to the versatility of the technique to use in screening binding events in order to save time, cost and obtained high data quality.