Search Results (4,380)

To analyze the phytogenic components of honey of Amorpha fruticosa L. (AFH) and establish a targeted quantitative method, the liquid chromatography-mass spectrometry (LC-MS) based metabolomic technology was used in this study. Firstly, high performance liquid chromatography—quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) untargeted metabolomics technology was used to screen candidate markers by comparing AFH metabolites with plant chemicals of Amorpha fruticosa L. Afterward, high performance liquid chromatography—triple quadrupole tandem mass spectrometry (HPLC-QQQ-MS/MS) was used to verify and identify the candidate markers, confirming ononin as the characteristic phytogenic marker of AFH, and determining its content range in AFH as 76.84–93.27 μg/kg (absent in acacia, rape, jujube, and Galla chinensis honey). Then, network pharmacology and molecular docking techniques were adopted to explore the gastric protective mechanism of ononin, and the results showed that ononin strongly binds AKT1 (binding free energy −8.0677 kcal/mol). Using the established method, the LC-MS analytical method for ononin in honey established in this study may be used for the authenticity identification of the characteristic phytogenic markers of AFH. Full article

Background/Objectives: Onion (Allium cepa) peems are an underutilized by-product rich in polyphenols. This study evaluated the physicochemical profile, and bioactive potential (antidiabetic, antimicrobial, antioxidant, and anticoagulant) of Moroccan red onion peels using integrated in vivo, in vitro, and in silico approaches. Methods: Moisture, pH, ash content, and mineral elements were determined, followed by phytochemical screening and three extractions: decoction E0, aqueous Soxhlet E1, and hydroethanolic Soxhlet E2 (70/30; ethanol/water, v/v). The measurement of polyphenols, flavonoids, and tannins was carried out using colorimetric methods, while the molecular profile was studied by high-performance liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry (HPLC/UV-ESI-MS). Biological activities were determined using 2,2-diphenyl-1-picrylhydrazyl, ferric reducing antioxidant power, and total antioxidant capacity assays (in vitro antioxidant); microdilution (antimicrobial); prothrombin time and activated partial thromboplastin time (anticoagulant); and α-amylase/α-glucosidase enzymatic inhibition and oral glucose tolerance tests on normoglycemic rats. Also, acute toxicity was evaluated, and molecular interactions between these proteins and ligands (docking, molecular dynamics, and MM-PBSA) were analyzed. Results: Physicochemical analyses showed an acidic pH (3.06) and high ash content (15.21%), with the concentration of regulated elements remaining within FAO/WHO limits. The extractive content was between 6.90% E0 and 19.18% E2. The E1 extract had the maximum amount of total polyphenols (178.95 mg GAE/g); on the other hand, E2 was the richest in flavonoids by 121.43 mg QE/g. The HPLC/ESI-MS analysis of E0 revealed 20 compounds, among which flavonoids (84.93%) were predominant, with isorhamnetin (30.26%), followed by quercetin and its glycosylated forms. E1 showed the most potent antioxidant effects (IC₅₀ DPPH, 22.38 µg/mL, as that of ascorbic acid). The antibacterial activity of E0 was especially potent towards Enterobacter cloacae and Pseudomonas aeruginosa (MIC 75 µg/mL). A mild dose-dependent anticoagulant effect was seen. Antidiabetic activity was found to be outstanding: α-amylase (IC₅₀ 62.75 µg/mL) and α-glucosidase (IC₅₀ 8.49 µg/mL, stronger than acarbose) inhibitions were corroborated in vivo by a considerable decrease in the glycemic area under the curve. The molecular docking study in silico demonstrated strong molecular interactions, especially for quercetin 4′-O-glucoside with good binding energies. Conclusions: A. cepa peels from Morocco can be considered a safe plant matrix containing bioactive flavonoids with strong antioxidant and selective antimicrobial activities and promising antidiabetic effects, supported by molecular modeling. Full article

The continuous emergence of New Psychoactive Substances (NPS) poses a significant challenge to public health and forensic toxicology due to their unpredictable pharmacology and rapid turnover on the illicit market. This study describes the development and validation of a high-resolution screening method for the simultaneous detection of 90 NPS in oral fluid (OF), a matrix of choice for non-invasive sampling and roadside testing. The analytical workflow utilizes a “dilute-and-shoot” approach (1:2 v/v dilution) followed by ultra-high-performance liquid chromatography coupled with a quadrupole-Orbitrap hybrid mass spectrometer (UHPLC-HRMS/MS). Chromatographic separation was achieved in 11 min using a biphenyl column and a gradient elution. The method was validated according to ANSI/ASB Standard 036 guidelines, covering 90 substances including synthetic cannabinoids (e.g., HHC, MDMB-4en-PINACA), synthetic cathinones, and high-risk synthetic opioids such as nitazenes and fentanyl analogues. Results showed high sensitivity, with limits of identification (LOI) reaching 1 ng/mL for 44.4% of the analytes and 5 ng/mL for 37.8%, while the remaining compounds showed higher LOIs ranging from 10 to 100 ng/mL. No significant matrix interference or carryover was observed. The method was successfully applied to real samples from external quality control programs and forensic cases. This robust and versatile screening tool is suitable for clinical and forensic applications, supporting the monitoring of emerging NPS trends. Full article

Styela plicata, an edible ascidian rich in diverse bioactive constituents, represents a promising source of marine natural products for therapeutic discovery. Here, bioactive components from a 95% ethanol extract of S. plicata (ESP) were characterized by HPLC-MS/MS, showing that the major constituents were oxygenated small molecules dominated by fatty acyls and carboxylic acid derivatives. In a mouse model of alcohol-induced liver injury, H-ESP treatment (300 mg/kg) significantly reduced serum levels of AST, ALT, and TG (p < 0.01), while effectively ameliorating pathological changes in liver tissue, reducing lipid accumulation and inflammatory responses. Transcriptome sequencing (H-ESP vs. model group) identified 1097 differentially expressed genes (172 upregulated and 925 downregulated), and KEGG analysis highlighted significant enrichment of the Toll-like receptor signaling pathway. ESP modulated hepatic metabolite expression, suppressed inflammation via TLR-4/NF-κB pathway inhibition, and boosted antioxidant defenses by activating Nrf2/HO-1 signaling, which was further confirmed by RT-qPCR and immunohistochemistry. ESP increased intestinal SCFAs (acetate, propionate, isobutyrate; p < 0.05), improved α-diversity and the Firmicutes/Bacteroidetes ratio, reversed shifts in Lactobacillus and Bifidobacterium, and partly restored Odoribacter, supporting a gut–liver axis mechanism. Overall, these findings indicate that ESP exerts hepatoprotective effects by modulating the gut–liver axis, and they provide insights for developing natural therapeutics against alcoholic liver disease. Full article

Exogenously induced RNA interference (exoRNAi) is a powerful biotechnology tool for precise gene regulation. The plant chalcone synthase (CHS) gene serves as a valuable model for molecular biology due to its central role in flavonoid biosynthesis. However, there are currently very few studies addressing the advantages and disadvantages of in vitro (enzymatic) or in vivo (bacterial) methods for producing double-stranded RNA (dsRNA) for exogenous application. This study aims to optimize and compare the two methods for producing dsRNAs targeting the Arabidopsis thaliana CHS gene: enzymatic synthesis in vitro using a commercial kit and bacterial synthesis in vivo using an engineered E. coli HT115 (DE3) system. Bacterial synthesis conditions were optimized with respect to IPTG concentration and cultivation time, and the resulting dsRNA preparations were purified and quality-controlled. Their biological activities were assessed by treating A. thaliana plants and analyzing the effects on AtCHS gene expression and flavonoid production using qRT-PCR and HPLC-MS. The results demonstrated that purified AtCHS-dsRNA from both methods effectively suppressed AtCHS expression and downstream flavonoid biosynthetic gene expression, leading to significant reductions in anthocyanins and flavanols. This study confirmed the efficacy of exogenous dsRNAs in regulating plant metabolic pathways and provided a comparative analysis of dsRNA synthesis methods, highlighting their benefits and limitations for practical applications in plant biology and protection. Full article

The herbal tea industry has experienced substantial growth, particularly regarding green tea (Camellia sinensis). In the manufacturing of filter tea, fine herbal dust is generated as a residual by-product during grinding and sieving and is typically discarded as waste. This study aims to explore the application of ultrasound-assisted extraction (UAE) for secondary valorisation of green tea herbal dust by investigating the effects of various parameters on extraction efficiency. Antiradical activity of UAE extracts was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and the total phenolic content (TPC) was measured using Folin–Ciocalteu’s assay. Furthermore, selected phenolics were quantified by HPLC and qualitatively characterised by liquid chromatography coupled with electrospray ionisation and quadrupole time-of-flight tandem mass spectrometry (LC-ESI-QToF-MS/MS). The results demonstrate that UAE parameters have a pronounced influence on the antioxidant activity, TPC, and individual polyphenolic profile of green tea herbal dust extracts. Ethanol–water mixtures at a ratio of around 40–60%, as well as moderate impulse regimes (around 60%) and extraction times (around 10 min), were the most suitable for extracting green tea polyphenols. Epigallocatechin gallate was the predominant phenolic component in most extracts, alongside epicatechin, epigallocatechin, catechin, and gallic acid. The findings highlight the UAE technique as a robust, green, and scalable method for valorising green tea by-products, thereby facilitating the development of high-value natural extracts for applications in the food, pharmaceutical, and cosmetic industries. Full article

The cell membrane serves as the first line of defense against adverse environmental factors and is first to adapt to changing conditions. Cell membranes in both coral and its symbionts, which use different membrane adaptation strategies, have to acclimatize to various abiotic stressors. As our molecular-genetics analysis showed, colonies of Junceella fragilis were associated with dinoflagellates Cladocopium thermophilum, Gerakladium endoclionum and Breviolum minutum. We analyzed the phospholipid (PL) molecular species of the wild and cultivated Junceella fragilis and their dinoflagellates (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), ceramideaminoethylphosphonate (CAEP)), as well as thylakoid membrane lipids of dinoflagellates (glycolipids and betaine lipids). When comparing wild and cultivated J. fragilis colonies, there were no significant differences in thylakoid lipids, but there were differences in host membrane phospholipids, namely in PC, PE and PS. Thus, the profile of PL molecular species of the membrane is very sensitive to environmental factors, which probably explains the observed differences in the profiles of molecular PL species in this study. Full article

The present study provides an exhaustive exploration of twelve macroalgal species from the Adriatic Sea, including seven brown algae (Ericaria amentacea, Fucus virsoides, Cutleria multifida, Cystoseira compressa, Cystoseira corniculata, Gongolaria barbata and Padina pavonica), three green algae (Codium adhaerens, Codium vermilara and Ulva lactuca), and two red algae (Scinaia furcellata and Asparagopsis taxiformis). Matrix solid-phase dispersion (MSPD) was applied as the extraction technique, using generally recognized as safe (GRAS) solvents. The bioactive profile of the extracts was assessed through the quantification of total phenolic content (TPC) and antioxidant activity. Among the three phyla, U. lactuca, F. virsoides and S. furcellata exhibited the highest TPC (0.8, 26 and 3.0 mgGAE·g⁻¹) and antioxidant activity (1.9, 38 and 7.5 mgTE·g⁻¹), respectively. Targeted HPLC-MS/MS analysis enabled the identification of nineteen phenolic compounds across all taxa. Chlorophyta showed a characteristic profile enriched in coumarins, benzaldehydes and flavanones, including the selective detection of 7-hydroxycoumarin in species with higher antioxidant potential. Additionally, compounds such as chlorogenic, rosmarinic and caffeic acids exhibited taxon-specific distributions that may explain differences in antioxidant activity. Complementary untargeted ultra-high performance liquid chromatography quadrupole time-of-flight (UHPLC-QToF) metabolomics analysis provided broader coverage, revealing eighty metabolites spanning phenolics, sugars, organic acids, lipids, amino acids and their derivatives. Notably, the proposed detection of fatty acid esters of hydroxy fatty acids (FAHFAs) represents the first report of these compounds in macroalgae, alongside a pronounced presence of sulphated phenolics. Overall, these findings provide a robust baseline on the bioactivity and chemical composition of Adriatic macroalgae, highlighting their value as a natural source of functional compounds. Full article

Global climate change poses a significant threat to viticulture, primarily due to high temperatures. This study examined temperature-induced changes in phenolic profiles in berries of three wine grape cultivars under 25, 35, and 45 °C for 0–48 h using HPLC-ESI-MS/MS. To investigate the molecular response to severe heat stress, transcriptomic analysis was conducted in Cabernet Sauvignon berries subjected to a 45 °C treatment. Results showed that the 45 °C treatment decreased the levels of anthocyanins (particularly delphinidin-3-O-glucoside and cyanidin-3-O-glucoside) in grape berries. Acylated anthocyanins, such as malvidin-3-O-(6-acetyl)-glucoside, exhibited enhanced stability at elevated temperatures. Additionally, high temperatures increased the levels of protocatechuic and gentisic acids and decreased those of rutin, ferulic acid, and p-coumaric acid. Transcriptomic and qRT-PCR analyses revealed that the expression of OMT, LDOX, GST, F3′5′H, and 3AT was positively correlated with changes in anthocyanin levels under high temperatures, suggesting their roles in the observed phenolic alterations. These findings highlight the molecular responses of winegrapes to heat stress, providing a foundation for future viticulture strategies under changing climatic conditions. Full article

The flowers of Sophora japonica L. (SJF) and Robinia pseudoacacia L. (RPF) are edible and similar in appearance. The chemical constituents of SJF and RPF were compared by UPLC-Q-TOF-MS/MS and HPLC analysis in this study. A total of 29 and 19 constituents were identified in SJF and RPF, respectively. Flavonoid glycosides were the main constituents found in both flowers. The main aglycon moieties found in SJF were quercetin, kaempferol and isorhamnetin, whereas acacetin and kaempferol were the main ones found in RPF. Additionally, the content of flavonoids in SJF was significantly higher than that in RPF, as determined by HPLC. Rutin was the most dominant flavonoid in SJF with a content range of 72.31~88.15 mg/g, followed by quercetin (13.05~20.30 mg/g). Kaempferol-di(rhamnoside)-hexoside was the most dominant flavonoid in RPF with a content range of 25.94~30.00 mg/g. The distinct flavonoid profiles indicated the chemical non-equivalence of SJF and RPF. Therefore, RPF should not be considered a direct substitute for SJF in herbal medicine without further pharmacological and clinical validation. Full article

Allium longistylum is a relatively understudied species whose phytochemical composition and biological activities remain largely unexplored. In this study, the first true leaf (FTL) and the second true leaf (STL) of A. longistylum were compared with respect to phenolic composition, antioxidant capacity, antimicrobial activity, and quorum-sensing (QS) inhibition. Total phenolic content (TPC) and total flavonoid content (TFC) were determined spectrophotometrically, while antioxidant activity was evaluated using ABTS and DPPH radical scavenging assays. Antimicrobial and anti-QS activities were assessed against Staphylococcus aureus, Acinetobacter baumannii, and Chromobacterium violaceum. STL exhibited significantly higher TPC and TFC than FTL, consistent with its stronger radical scavenging activity. Both extracts showed moderate antimicrobial activity and reduced violacein production in C. violaceum, indicating interference with QS. UPLC-Q-Orbitrap-ESI-MS/MS profiling tentatively identified several phenolic acids and flavonoid derivatives. HPLC analysis confirmed the presence of selected phenolic compounds, although several prominent peaks in the chromatograms remained unidentified. Many of the compounds detected by UPLC-Q-Orbitrap-ESI-MS/MS and HPLC have previously been reported to exhibit antioxidant, antimicrobial, and anti-QS activities; their presence may therefore contribute to the bioactivities observed in both extracts. However, their contribution to the observed effects remains speculative and requires further validation through targeted isolation and bioactivity testing. The results suggest that A. longistylum is a promising source of phenolic compounds with antioxidant and antimicrobial properties. Full article

Momordica balsamina Linn. is widely used in traditional medicine for the management of diabetes; however, the specific bioactive compounds responsible for this activity have not been fully isolated and structurally elucidated from South African populations. This study reports, for the first time, the isolation and comprehensive characterization of antidiabetic compounds from South African samples of M. balsamina. Crude extracts were obtained through sequential solvent extraction, followed by isolation and purification using vacuum liquid chromatography. Structural elucidation was achieved using HPLC, UPLC–MS, FTIR, and NMR spectroscopy. The antidiabetic potential of the isolated compounds was evaluated through inhibition assays against α-amylase, α-glucosidase, and β-glucosidase. Molecular docking was employed to examine binding interactions with these target enzymes, while cytotoxicity was assessed using the MTT assay against Vero and HEK-293 cell lines. Two compounds, DD26.27 and EAEA1.2, were successfully isolated from dichloromethane and ethyl acetate extracts, respectively. Both compounds demonstrated concentration-dependent inhibition of the tested enzymes. Notably, molecular docking revealed strong binding affinities and favorable interactions with key catalytic residues, surpassing the standard drug acarbose and corroborating the in vitro results. Cytotoxicity studies confirmed that, at lower concentrations, the compounds were non-toxic to the tested cell lines. Collectively, these findings provide novel scientific validation of the traditional use of M. balsamina in South Africa and identify promising lead compounds for further in vivo studies and antidiabetic drug development targeting postprandial hyperglycemia. Full article

Background: B7-H3, a type I transmembrane glycoprotein belonging to the B7 superfamily, is an attractive target for antitumor therapies. B7-H3 demonstrates aberrant overexpression in various types of solid tumors while showing limited and low expression in normal human organs. Various types of treatment targeting B7-H3 have been reported. Among these treatments, antibody–drug conjugates (ADCs) have shown potent activity, and several clinical trials, including DS7300a and MGC018, are currently ongoing. Methods: Here, we constructed CD276-8 ADC, composed of the anti-B7-H3 antibody CD276-8 with moderate affinity, an enzymatically cleavable tetra-peptide-based linker and DXd. Characteristics, including in vitro binding affinity and internalization activity, were assessed by bio-layer interferometry (BLI), flow cytometry and high content analysis (HCA). The cytotoxicity of CD276-8 ADC was evaluated in cell lines expressing B7-H3. Pharmacokinetic profiles and antitumor activity were evaluated in mouse models in vivo. Finally, the developability of CD276-8 ADC was assessed with plasma stability, accelerated stability and freeze–thaw studies using LC-MS and HPLC. Results: Characterization in vitro demonstrated the moderate affinity and acceptable internalization activity of CD276-8 ADC. In addition, CD276-8 ADC exhibited potent antitumor activities in B7-H3-positive cell line-derived xenograft (CDX) models with acceptable pharmacokinetic profiles, although it showed less potent cytotoxicity in various cell lines in vitro, indicating acceptable developability. Conclusions: We developed CD276-8 ADC, a B7-H3-targeting ADC with moderate affinity, which delivers the TOP1 inhibitor DXd. This design combined moderate affinity and acceptable pharmacokinetics, resulting in potent antitumor efficacy in vivo. Our study suggests that affinity optimization could be a useful consideration for enhancing ADC efficacy, positioning CD276-8 ADC as a promising therapeutic for B7-H3-expressing solid tumors. Full article

In this study, an ion-pair reverse-phase high-performance liquid chromatography–electrospray ionization mass spectrometry (RP-HPLC-ESI-MS) method was optimized and validated for purity determination for the quality control of the proposed generic nusinersen oligonucleotide drug substance and drug product. The optimization and considerations of sample preparation, chromatographic and mass spectrometry conditions are discussed. The limit of detection was 2.5 × 10⁻⁵ mg/mL and the limit of quantitation was 4.9 × 10⁻⁵ mg/mL. The linearity of the signal (XIC) for all impurities was linear with correlation coefficients of R² ≥ 0.9669. This study, associated with the development of therapeutic oligonucleotides, examines the subject of product-related impurities. The authors consider an ion-pair reverse-phase high-performance liquid chromatography in combination with mass spectrometry for impurity quantitative control. This study contributes to the field by elucidating several critical aspects that, while previously unaddressed in the existing literature, are essential for developing effective analytical methods. Full article

Human infertility affects approximately 17.5% of the global population, with male factors accounting for nearly half of all cases. Identifying reliable molecular biomarkers is crucial for improving the diagnosis and assessment of male fertility. This study established and refined an untargeted high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC-ESI-MS/MS) protocol for a comprehensive lipidomic and metabolomic analysis of human spermatozoa, using only 1.25 million cells per sample. Compared with previous reports, our optimized method achieved an unparalleled level of analytical depth, identifying 473 lipid species and 955 structurally annotated metabolites. This corresponds to nearly a 7600-fold improvement in detection efficiency per cell compared with previously published approaches. Lipidomic analysis revealed that the most abundant lipid classes were glycerophospholipids (39%), cholesterol (20%) and fatty acids (19%), with cholesterol representing the single most abundant compound. This observation is consistent with the structural complexity of the sperm plasma membrane. Metabolomic profiling similarly identified glycerophospholipids (44%), eicosanoids (14%) and N-acyl amino acids (12%) as the major metabolite classes. The integration of lipidomic and metabolomic data highlighted functionally interconnected pathways related to membrane dynamics, energy metabolism, and hormone biosynthesis. Overall, this work establishes a robust, sensitive, and scalable analytical framework that enables the high-coverage molecular characterization of spermatozoa from limited sample material, laying the groundwork for future biomarker discovery and clinical applications in male infertility research. Full article