Search Results (861)

The aim of this study was to evaluate, on a preliminary basis, the ability of multivariate techniques to predict the antioxidant activity of selected Stachys and Betonica species, based on chromatographic data. The methanol extracts of six Stachys species and ten Betonica species were analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain their chromatographic profiles. The phytochemical similarity of the samples was assessed using a selected chemometric method (principal component analysis (PCA) and hierarchical cluster analysis (HCA)). The antioxidant activity of the studied extracts (DPPH with 2,2-diphenyl-1-picrylhydrazyl reagent and FRAP—ferric reducing antioxidant power) was determined using a spectrophotometric technique. A multivariate PLS model was then used to predict the antioxidant activity of the methanolic extracts of Stachys and Betonica species based on their RP-HPLC fingerprints. The two obtained PLS models proved useful for predicting the biological activity of the tested extracts. High correlation coefficients (DPPH: R² = 0.9963; FRAP: R² = 0.9895) confirmed the reliability of the PLS prediction model. The results confirmed the effectiveness of combining qualitative and quantitative chromatographic fingerprinting methods with antioxidant activity testing and chemometric analysis, demonstrating that extracts from Stachys and Betonica are a rich source of bioactive substances with antioxidant properties. Full article

The simultaneous HPLC method for quantifying Quercetin (Que), Thymoquinone (Thy), and Pterostilbene (Pte) aims at the precise measurement of these polyphenols alone or in complex mixtures, targeting their therapeutic potential in disorders such as diabetes and epilepsy. The method focuses on quantifying Que, Thy, and Pte, utilizing optimized reversed-phase HPLC conditions as per ICH Q2(R1) standards. Key validation aspects include linearity, specificity, precision, and accuracy, ensuring compliance for quality control in nanomedicine and nutraceuticals, and the method’s applications support pharmacokinetic studies and stability testing, contributing to personalized medicine and addressing pharmaco-resistance. The HPLC method development and validation were performed on a phenyl column using the mobile phase consisting of solvent A (0.1% orthophosphoric acid in HPLC water) and solvent B (acetonitrile) at a ratio of 55:45 in an isocratic elution mode at a flow rate of 1 mL/min and at a column temperature of 35 °C. Ultraviolet detection was measured at 254 nm. Moreover, the method was validated for accuracy, precision, linearity, specificity, and sensitivity. The retention time for tested Que, Thy, and Pte was observed at 4.15 min, 8.70 min, and 10.75 min, respectively. Limits of detection for Que, Thy, and Pte were 1.55 μg/mL, 2.40 μg/mL, and 70.79 µg/mL, whereas limits of quantification were 4.69 μg/mL, 7.28 μg/mL, and 214.52 µg/mL, respectively. Linearity and correlation coefficients for Que, Thy, and Pte were found in the range of 50–250 μg/mL (0.9999), 50–250 μg/mL (0.9999), and 620–3100 μg/mL (0.9996), respectively. A reasonable level of accuracy was indicated by the tested method suggesting extremely high recovery levels (98–102%). The separation of tested compounds was achieved within 11 min. The developed and validated RP-HPLC–UV method was successfully applied for the identification and quantification of Que, Thy, and Pte for their combined estimation in complex formulations. From the validation study, it was found that the tested method is specific, accurate, precise, reliable, and reproducible. Full article

The use of lecithin as an emulsifier in food supplements has increased in recent years. However, successful formation of liposomes or micelles requires an appropriate mixture of phospholipids in lecithin. To evaluate the emulsification properties of lecithin for food supplements, a reliable analytical procedure for characterizing phospholipids is necessary. A liquid chromatography–mass spectrometry method was developed to identify phospholipids in lecithin without standard reference materials. For efficient separation of phospholipids before mass spectrometric analysis, a reverse-phase high-performance liquid chromatography method was optimized using a Waters XBridge Protein BEH C4 column. The optimized chromatographic method demonstrated good linearity and precision. Molecular ions were detected in full scan mode to determine accurate mass-to-charge ratios for individual peaks in the chromatogram. A custom Python program was then used to generate a list of possible phospholipid species for each peak based on the measured mass-to-charge ratios. Tandem mass spectrometry was performed to confirm the identity of specific phospholipids by comparing experimental fragmentation patterns with theoretical predictions. Identification of the phospholipids was also confirmed with four commercially available standard reference compounds, demonstrating the reliability of the proposed approach. The developed method offers a practical and cost-effective strategy for identifying phospholipids in complex matrices, especially when standard reference compounds are unavailable. Additionally, it enables targeted selection of standard compounds for future quantitative analyses, making it a valuable tool for comprehensive lipid profiling. Full article

Reversed-phase high-performance liquid chromatography (RP-HPLC) remains one of the most widely applied analytical techniques in the development and quality control testing of finished pharmaceutical products. The combination of gradient chromatographic methods with diode-array detection (DAD) enhances selectivity, ensuring accuracy and reliability when testing drugs with diverse chemical properties in a single dosage form (i.e., fixed-dose combination (FDC) products). In this study, an RP-DAD-HPLC method was developed for the quantitative analysis of clofazimine (CFZ) and pyrazinamide (PZA) for inclusion in an FDC topical drug delivery system. Chromatographic separation was achieved using a C18 column (4.6 mm × 150 mm, 5 µm particle size) with gradient elution at 1 mL/min, employing 0.1% aqueous formic acid and acetonitrile (mobile phases). PZA and CFZ were detected at 254 nm and 284 nm, respectively. The method was validated in accordance with ICH Q2 guidelines, assessing specificity (considering interference from solvents, product matrix, and degradation products), linearity (7.8–500.0 µg/mL, r² = 0.9999), system repeatability (%RSD ≤ 2.7%), and intermediate precision (25–500 µg/mL, %RSD ≤ 0.85%). Method robustness was evaluated using a three-level Box–Behnken design (BBD) with response surface methodology (RSM) to assess the effects of variations in detection wavelength, mobile phase flow rate, and column temperature. Full article

The saw-scaled viper Echis carinatus, one of the “Big Four” causes of snakebites in India, is found from Sri Lanka to eastern Iraq. To investigate clinical reports regarding the limited efficacy of Indian polyvalent antivenom (IPAV) against envenomation in Echis carinatus sochureki (ECS) in northwestern India, we obtained 22 snakes from three locations in Rajasthan and identified 148–174 toxin isoforms belonging to 21–25 toxin families in their venom using a bottom-up proteomics approach. All samples showed a high abundance of snake venom metalloproteinases (SVMPs), particularly SVMP class III. Other major components were phospholipases A₂, L-amino-acid oxidases, snake venom serine proteases and snaclecs (C-type lectins). Variation in venom composition among locations in Rajasthan, compared to E. c. carinatus (ECC) from southern India, was primarily due to differences in the relative abundance of these toxin families. Recognition of all venom components by IPAV was poor at lower antivenom concentrations. Notably, SVMP classes II and III were poorly recognized at all venom-to-antivenom ratios in all ECS venoms, and a plasma clotting assay revealed poor neutralization of procoagulant activity. This collaborative study highlights the need for the development of regional antivenoms to effectively treat snakebites in northwestern India. Full article

Dendropanax morbifera (DM; “Hwangchil”) is an evergreen tree native to southern Korea and Jeju Island, traditionally used for detoxification, anti-inflammatory, immunomodulatory, and neuroprotective purposes. Recent studies indicate that DM extracts and their constituents exhibit a broad range of biological activities, including antioxidant, anti-inflammatory, antimicrobial, anticancer, antidiabetic, hepatoprotective, and neuroprotective effects. Phytochemical investigations have revealed a chemically diverse profile comprising phenolic acids, flavonoids, diterpenoids, triterpenoids—most notably dendropanoxide—and polyacetylenes, with marked variation in compound distribution across plant parts. Despite this progress, translational application remains constrained by the lack of standardized extraction protocols, substantial variability in high-performance liquid chromatography (HPLC) methodologies, and limited mechanistic validation of reported bioactivities. This review proposes an integrated framework that links extraction strategies tailored to compound class and plant part with standardized C18 reverse-phase HPLC conditions to enhance analytical reproducibility. In parallel, in silico target prediction using SwissTargetPrediction is applied as a hypothesis-generating approach to prioritize potential molecular targets for subsequent experimental validation. By emphasizing methodological harmonization, critical evaluation of evidence levels, and systems-level consideration of multi-compound interactions, this review aims to clarify structure–activity relationships, support pharmacokinetic and safety assessment, and facilitate the rational development of DM-derived materials for medical, nutritional, and cosmetic applications. Full article

Background: Uncontrolled diabetes is characterised by a loss of blood glucose control and increased oxidation of fatty acids to produce ATP. Use of metabolic inhibitors to blunt fatty acid oxidation and restore glucose metabolism is a poorly studied intervention for diabetes. Methods: Steptozotocin-induced diabetes was developed in Wistar male rats. A subset was supplemented with mildronate (100 mg/kg—14 days). Exploiting liquid chromatography-mass spectrometry for workflows including ion exchange-, C18-reverse phase- and HILIC-based chromatography methods, metabolite levels were quantified in plasma liver and brain tissue. Using both untargeted and targeted metabolomic analysis changes to the global tissue metabolome and individual metabolic pathways were estimated. Results: We document that an inhibitor of carnitine synthesis, mildronate, decreased plasma (50% p < 0.01) carnitine abundance and decreased plasma glucose concentration by one-third compared to streptozotocin (STZ)-treated rats (p < 0.001). Targeted metabolomic analysis of the liver showed decreased alpha-ketoglutarate abundance (35% p < 0.05) by STZ diabetes that was further decreased following mildronate treatment (50% p < 0.05). For both beta-hydroxybutyrate and succinate levels, STZ diabetes increased hepatic abundance by 50% (p < 0.05 for both), which was restored to control levels by mildronate (p < 0.05 for both). In contrast, brain TCA intermediate abundances were unaffected by either STZ diabetes or mildronate (NS for all). STZ diabetes also decreased abundance of pentose phosphate pathway (PPP) metabolites in the liver (glucose-6-phosphate, 6-phosphogluconolactone, 6-phosphogluconate 50% for all; p < 0.05), which was not restored by mildronate treatment. However, brain PPP metabolite abundance was unchanged by STZ diabetes or mildronate (NS for all). However, mildronate treatment did not affect the increased abundance of brain sorbitol, sorbitol-6-phosphate and glucose-6-phosphate as a result of STZ diabetes. Conclusions: Together, these observations highlight the potential role that metabolic inhibitors, like mildronate, may play in restoring blood glucose for diabetic patients, without a direct effect of tissues that represent obligate consumers of glucose (e.g., brain) whilst manipulating fat oxidation in tissues such as the liver. Full article

Dipeptidyl peptidase-4 (DPP-4) is a protease that degrades incretin and inhibits the secretion of insulin. Consequently, DPP-4 inhibition promotes insulin secretion and prevents the onset of type 2 diabetes. Given the growing interest in food-derived DPP-4 inhibitory peptides as potential functional ingredients, buckwheat (Fagopyrum esculentum) represents a promising source; however, few studies have investigated the bioactivity of peptides derived from buckwheat flour hydrolysates. In this study, two DPP-4 inhibitory peptides, Ile-Pro-Trp and Ile-Pro-Leu, were identified through purification of buckwheat flour hydrolysate and liquid chromatography–tandem mass spectrometry analysis. In a rat oral glucose tolerance test (OGTT), a fraction of buckwheat flour hydrolysate, crudely purified by reverse-phase column chromatography, showed a non-significant trend toward reducing increases in blood glucose. To our knowledge, this study is the first to show that Ile-Pro-Trp isolated from food protein hydrolysates exhibits considerable DPP-4 inhibitory activity. Moreover, this is the first study identifying Ile-Pro-Ile as a DPP-4 inhibitor from a plant source. Full article

Biogenic amines (BAs) can be found in various foods, such as cheese, wine, and chocolate. The consumption of a sufficient quantity of BA can lead to symptoms including headaches, hypertonia, and diarrhea. For this reason, the amount of BA in food is regulated in many countries. A new method for the determination of biogenic amines in wine has been proposed, which involves derivatizing BA with p-toluene sulfonyl chloride (TsCl) and using K₂S₂O₈ to reduce the matrix effect. The derivatives of putrescine, cadaverine, histamine, and tyramine with TsCl were synthesized and characterized by ¹H NMR spectroscopy. Separation of BA derivatives was performed using a reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic system was equipped with a reversed-phase C8 column and a diode array detector. This method was validated to analyze the above-mentioned biogenic amines simultaneously in red and white wine samples. The detection limits for putrescine, cadaverine, histamine, and tyramine in wine samples were 0.0248 mg·L⁻¹, 0.0645 mg·L⁻¹, 0.346 mg·L⁻¹ and 0.00866 mg·L⁻¹, respectively. The calibration curves showed good linearity (r > 0.999), and biogenic amines recovery varied from 83.0 to 110%. The proposed method demonstrates high sensitivity, straightforward sample preparation, and rapid analysis time. Full article

Selenium-enriched oyster proteins were hydrolyzed using trypsin to obtain peptides with angiotensin-I-converting enzyme (ACE) inhibitory activity. The hydrolysate was purified by ultrafiltration and two-step reversed-phase high-performance liquid chromatography (RP-HPLC), yielding the most active fraction M4-2 (selenium content: 37.00 ± 0.56 mg/kg; IC₅₀: 0.774 mg/mL, significantly lower than the IC₅₀ of the crude hydrolysate, 2.801 mg/mL). This fraction was further analyzed by LC-MS/MS and molecular docking, leading to the identification of 91 selenium-containing peptide sequences. Two novel peptides, SeMFRTSSK and QASeMNEATGGK, showing strong binding affinities (−9.8 and −9.0 kcal/mol, respectively), were selected. Molecular docking revealed that SeMFRTSSK bound to key residues in the ACE active pocket via hydrogen bonds, whereas QASeMNEATGGK interacted with the Zn²⁺ active center. Cellular assays using EA.hy926 cells demonstrated that both peptides were non-cytotoxic at concentrations up to 0.25 mg/mL. At 0.025 mg/mL, SeMFRTSSK and QASeMNEATGGK enhanced cellular NO release by 202.65% and 273.45%, respectively, while suppressing Endothelin-1 (ET-1) secretion by 18.03% and 27.86%, compared to the blank control group. Notably, these peptides induced higher levels of NO release and greater suppression of ET-1 secretion than those in the captopril-treated positive control group. These findings support selenium-enriched oyster-derived peptides as potential natural antihypertensive ingredients. Full article

A robust and stability-indicating Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the quantitative determination of bexagliflozin and its related impurities in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH Q2(R1)) guidelines. Chromatographic separation was achieved on a C18 column using a mobile phase of methanol and ammonium acetate buffer (pH 4.2) in a 60:40 (v/v) ratio, with a flow rate of 1.0 mL·min⁻¹ and UV detection at 220 nm. The method was validated for linearity, sensitivity (LOD and LOQ), precision, robustness, and system suitability, all within acceptable limits for low-concentration analysis. Excellent linearity (r² > 0.999) and precision (%RSD 0.3–4.4%) confirmed its reliability for stability assessment. The assay was performed at 100 µg·mL⁻¹, where all validation parameters showed %RSD values ≤ 2%, demonstrating high precision and robustness. Forced degradation studies under acidic, basic, oxidative, photolytic, and thermal conditions revealed a major degradation product formed under acidic stress. This product was isolated and structurally characterized using LC–MS, ¹H NMR, and ¹³C NMR, and is reported here for the first time. The proposed RP-HPLC method proved to be specific, precise, and reliable for the determination of bexagliflozin and its related impurities, making it suitable for routine stability testing, quality control, and pharmaceutical development applications. Full article

Many polar compounds of biochemical and pharmaceutical relevance exhibit low retention in reversed-phase liquid chromatography (RPLC), making their separation challenging. While hydrophilic interaction liquid chromatography (HILIC) columns are commonly used for such analyses, they require mobile phases with high organic solvent content. This work explores an alternative approach using RPLC with conventional C18 columns and mobile phases containing low percentages of acetonitrile, along with small amounts of the surfactant sodium dodecyl sulfate (SDS). This combination significantly enhances the retention of highly polar compounds. When the SDS concentration is sufficiently low, below the critical micellar concentration in water (8 mM), the retention increase follows a linear pattern. The retention behavior of polar compounds with different properties (nucleosides, methylxanthines, sulfonamides, and the diuretic hydrochlorothiazide) is examined using mobile phases in the submicellar region, with SDS concentrations ranging from 0 to 0.3 mM, acetonitrile contents between 10 and 20% (v/v), and temperatures varying from 25 to 55 °C. Changes in peak half-widths are also analyzed. Since SDS adsorbs onto the stationary phase, modifying its surface, the equilibration time has been investigated as a critical factor affecting retention reproducibility, influenced by the SDS concentration, acetonitrile content, and temperature. The results emphasize the need for complete equilibration to ensure reliable and consistent results. Full article

Oleanolic acid (OA) is a natural pentacyclic triterpenoid with reported hypoglycemic, hepatoprotective, antidiabetic, and anti-inflammatory activities. However, its limited aqueous solubility restricts its formulation and potential biomedical applications. To address this limitation, we designed a hydrophilic OA derivative, 1a, by introducing an amino acid fragment at the C-28 position. We established a reverse-phase high-performance liquid chromatography (HPLC)-based method to measure the octanol–water partition coefficient (Log P_ow) of OA and 1a. Under neutral conditions, 1a showed a markedly reduced Log P_ow value (2.91 ± 0.02) compared with OA (4.30 ± 0.01), confirming substantially improved hydrophilicity. The biological compatibility of OA and 1a was further evaluated using in vitro human islet cultures. Both compounds maintained high islet viability (approximately 90%). In addition, islets pre-treated with 1a exhibited viability, purity, and insulin expression levels comparable to those observed with OA treatment, indicating that the C-28 modification preserved OA’s biological properties while improving solubility. Overall, this proof-of-concept study demonstrates that C-28 amino-functionalization can improve the physicochemical properties of OA without compromising its compatibility with human islets. The HPLC-based Log P_ow method established here provides a practical analytical tool for future structure–activity investigations of OA derivatives, and the improved solubility of 1a may facilitate its use in human islet preparation workflows. Full article

Background/Objectives: Previously reported analyses show that some androstane-3-oxime derivatives with picolyl and picolinylidene functional groups possess a significant anticancer activity towards various cancer cell lines. These findings suggest that these compounds have prominent biological potential and represent a good basis for further research. The present study aims to determine their anisotropic lipophilicity as a physicochemical parameter relevant to both prediction of chromatographic behavior and biological activity. Methods: Anisotropic lipophilicity was determined using reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) systems equipped with three stationary phases (C18, C8 and phenyl) and three mobile phases composed of water with different modifiers (methanol, acetonitrile and a methanol-acetonitrile mixture). Capacity factors (logk) were obtained for all compounds across the chromatographic systems to describe their behavior in anisotropic environments. Chemometric analyses were performed using linear pattern recognition techniques (PRT), such as hierarchical cluster analysis (HCA) and principal component analysis (PCA), and non-linear clustering based on artificial neural networks (CANN). Results: The experimentally determined chromatographic parameters were correlated with in silico lipophilicity values (logP). This comparison allowed for examination of the concordance between experimental and computed data for the series of androstanes. The chemometric analysis resulted in models that provided an overview of the grouping of compounds in the space of the determined chromatographic parameters. Conclusions: The results demonstrate strong agreement between experimental and computational lipophilicity parameters. This very good data fit provides a reliable foundation for further studies exploring the relationships between lipophilicity and biological activity of the studied androstane derivatives. Full article

Iron (Fe) chelators are used to treat iron-overloaded disorders, metal detoxification, radionuclides, and molecular imaging; however, they can cause side effects. In this study, we identified and characterized Coprogen B (CPGB), a hexadentate trihydroxamate siderophore secreted by the opportunistic dimorphic fungus Talaromyces marneffei and compared its properties with deferoxamine (DFO). Siderophore production was enriched from a ΔsreA strain and purified via Amberlite XAD2 and Sephadex LH20 chromatography, followed by reverse-phase HPLC. Active fractions were confirmed by Ultraviolet–Visible (UV–Vis) spectral fingerprints (≈230 nm) for hydroxamate, with a band at 430–450 nm upon Fe(III) complexation, as well as by chrome azurol A assay, Nuclear Magnetic Resonane (NMR) spectroscopy, High-Performance Liquid Chromatography–Mass Spectrometry (HPLC-MS), and Matrix-Assisted Laser Desorption/Ionization–Time-of-Flight Mass Spectrometry (MALDI-TOF-MS). CPGB exhibited strong molar absorptivity and rapid, concentration-dependent chelation of Fe(III), yielding a sustained binding profile that matched or exceeded that of DFO over time. In determining n-octanol/water partitioning for CPGB and DFO (230 nm) and their Fe(III) complexes, the partitioning (P) assay revealed that CPGB was moderately hydrophilic (P = 0.505 ± 0.063; cLogP = −0.299 ± 0.053), while DFO was strongly hydrophilic (P = 0.098 ± 0.005; cLogP = −1.010 ± 0.022). Fe(III) complexation reduced lipophilicity: CPGB–Fe partitioned ~30–35% into octanol, while DFO–Fe complex partitioned ~7–8%, remaining largely aqueous. Overall, this outcome potentially suggested improved clearance in vivo. These data nominate CPGB as a promising alternative to existing iron chelators. The siderophore exhibited greater lipophilicity, emphasizing better passive membrane permeability than DFO, while siderophore–Fe(III) binding indicated increased biases toward the aqueous phase. Future in vivo studies are warranted to confirm its pharmacokinetics, safety, and therapeutic efficacy. Full article