Evidence to support cultivated fruiting body of Ophiocordyceps sinensis (Ascomycota)'s role in relaxing airway smooth muscle.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiocordyceps sinensis (O. sinensis) is a genus of Ascomycete fungus that is endemic to the alpine meadows of the Tibetan Plateau and adjoining Himalayas. It has been used traditionally as a tonic to improve respiratory health in ancient China as well as to promote vitality and longevity. Bioactive components found in O. sinensis such as adenosine, cordycepin, 3-deoxyadenosine, L-arginine and polysaccharides have gained increasing interest in recent years due to their antioxidative and other properties, which include anti-asthmatic, antiviral, immunomodulation and improvement of general health. AIM OF THE STUDY: This study's primary aim was to investigate the effect of a cultivated fruiting body of O. sinensis strain (OCS02®) on airways patency and the secondary focus was to investigate its effect on the lifespan of Caenorhabditis elegans. MATERIALS AND METHODS: A cultivated strain, OCS02®, was employed and the metabolic profile of its cold-water extract (CWE) was analysed through liquid chromatography-mass spectrometry (LC-MS). Organ bath approach was used to investigate the pharmacological properties of OCS02® CWE when applied on airway tissues obtained from adult male Sprague-Dawley rats. The airway relaxation mechanisms of OCS02® CWE were explored using pharmacological tools, where the key regulators in airway relaxation and constriction were investigated. For the longevity study, age-synchronised, pos-1 RNAi-treated wild-type type Caenorhabditis elegans at the L4 stage were utilised for a lifespan assay. RESULTS: Various glycopeptides and amino acids, particularly a high concentration of L-arginine, were identified from the LC-MS analysis. In airway tissues, OCS02® CWE induced a significantly greater concentration-dependent relaxation when compared to salbutamol. The relaxation response was significantly attenuated in the presence of NG-Nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) and several K+ channel blockers. The longevity effect induced by OCS02® CWE (5 mg/mL and above) was observed in C. elegans by at least 17%. CONCLUSIONS: These findings suggest that the airway relaxation mechanisms of OCS02® CWE involved cGMP-dependent and cGMP-independent nitric oxide signalling pathways. This study provides evidence that the cultivated strain of OCS02® exhibits airway relaxation effects which supports the traditional use of its wild O. sinensis in strengthening respiratory health.

Rapid Characterization of Phospholipids from Biological Matrix Enabled by Indium Tin Oxide (ITO) Coated Slide Assisted Enrichment MALDI Mass Spectrometry.

Lipidomic analysis of human serum is essential to monitor the individual's health status. Herein, we develop a facile strategy for rapid characterization of phospholipids in human serum via indium tin oxide (ITO) coated glass slide solid phase extraction MALDI mass spectrometry (ITO-SPE-MALDI-MS). Phospholipid species are retained on ITO slide via solid phase extraction owing to the unique property of the ITO material; the measurement of phospholipid species from 1 µl human serum within 2 min is achievable. A comparison of ITO-SPE strategy with conventional extraction methods was further carried out using liquid chromatography-mass spectrometry (LC-MS) and ion-mobility mass spectrometry (IM-MS), resulting in a comparable enrichment performance for the phospholipid analysis. Furthermore, rapid lipidomic profiling of serum samples from human colorectal cancer patients and cell lines was demonstrated. Our results indicate that ITO-SPE-MALDI-MS provides a higher throughput strategy for the analysis of phospholipid species in complex biological mixtures, showcasing its potential for applications in the analysis of clinical biofluids.

Effects of Different Manufacturing Processes on Thermal and Photo-oxidative Stability of Sesame Oil.

The roasting process of sesame oil is expected to alter its internal composition and stability under oxidation condition. Presumably, the effect of roasting may differ with oxidation conditions (i.e., thermal and photo-oxidation), but such studies have not been undertaken. To further evaluate this notion, several type of sesame oils (raw and refined as unroasted oil, and roasted oil) and rapeseed oils as comparison were subjected to thermal oxidation (120℃) and photo-oxidation (50,000 lx) for 5 and 10 hours. The result revealed that the roasting sesame oil exhibited good stability under thermal oxidation, possibly due to the change on antioxidant agents such as sesamol and Maillard products during the roasting process. In contrast, the refined sesame oil (unroasted) demonstrated high stability under photo-oxidation, indicating that the refining process has a more significant impact on the oxidative stability in sesame oil compared to the alterations in its components caused by the roasting process. Taken together, this study is the first to show that the roasting and refining processes of sesame oil alter its internal composition and show different variations in sesame oils' oxidative stability under thermal and photo-oxidation, which holds significance considering its global consumption.

Comprehensive phenolic profiling and biological evaluation of Centaurea glastifolia L. (Asteraceae).

The present investigation focused on the comprehensive analysis of the phenolic profile of Centaurea glastifolia L. (Asteraceae) and the assessment of its diverse biological activities. Utilising LC-MS/MS, the phytochemical composition of the 70% methanol extract of Centaurea glastifolia (CG-ME) was thoroughly elucidated, revealing the presence of 30 distinct phytochemical compounds. Notably, major phenolic constituents identified in the extract included quinic acid, chlorogenic acid, luteolin-7-O-glucoside, kaempferol-3-O-glucoside, luteolin, and apigenin-7-O-glucoside. The antioxidant, antibacterial, antiproliferative, and cytotoxic activities of CG-ME were investigated. The CG-ME exhibited a moderate capacity for scavenging DPPH radicals (IC50: 50.05 ± 1.58 µg/mL) and FRAP (63.96 ± 0.39 mg TE/g extract), indicating a moderate level of antioxidant activity. Moreover, CG-ME demonstrated significant antiproliferative effects (GI50: 1.10 and 1.30 µg/mL) on cancer cells (C6 and HTC cancer cell lines, respectively) while displaying low cytotoxicity towards normal cells (LC50: >1000 µg/mL). In terms of antibacterial activity, CG-ME was found to be inactive against tested both Gram-positive and Gram-negative bacterial strains (MIC > 500 µg/mL). The extracts had a promising antiproliferative effect on C6, HeLa, and HT29 cancer cell lines with a less cytotoxic effect (10.5-14.2%) against normal cells.

A Comparative Study of LC-MS and FIA-(ESI)MS for Quantitation of S-Allyl-L-Cysteine in Aged Garlic Supplements.

The increasing consumption of food supplements demands the development of improved analytical methodologies to ensure their quality and authenticity. In this paper, two new approaches, liquid chromatography coupled to mass spectrometry (LC-MS) and flow injection analysis-(electrospray ionization) mass spectrometry (FIA-(ESI)MS), were optimized and validated for their application in the quantitative analysis of bioactive S-allyl-L-cysteine (SAC) in commercial aged garlic supplements (AGS). Although both methodologies were found to be useful for the sensitive and precise quantitation of SAC, the LC-MS approach allowed the differential determination of SAC and its bioactive diastereoisomer, S-1-propenyl-L-cysteine (S1PC), together with the identification of a number of organosulfur compounds typical of garlic. Mass fingerprints by FIA-(ESI)MS were proposed as an advantageous alternative to LC-MS analysis when the fast (4 min/sample) screening of AGS for their SAC content is intended, as in applications aimed at high-throughput quality control or standardization. Finally, the results gathered by the application of these two methodologies evidenced the highly variable composition of commercial AGS, as well as the identification of a number of potential composition frauds affecting their genuineness and benefits on health.

Crucial Parameters for Immunopeptidome Characterization: A Systematic Evaluation.

Immunopeptidomics is the area of knowledge focused on the study of peptides assembled in the major histocompatibility complex (MHC), or human leukocyte antigen (HLA) in humans, which could activate the immune response via specific and selective T cell recognition. Advances in high-sensitivity mass spectrometry have enabled the detailed identification and quantification of the immunopeptidome, significantly impacting fields like oncology, infections, and autoimmune diseases. Current immunopeptidomics approaches primarily focus on workflows to identify immunopeptides from HLA molecules, requiring the isolation of the HLA from relevant cells or tissues. Common critical steps in these workflows, such as cell lysis, HLA immunoenrichment, and peptide isolation, significantly influence outcomes. A systematic evaluation of these steps led to the creation of an 'Immunopeptidome Score' to enhance the reproducibility and robustness of these workflows. This score, derived from LC-MS/MS datasets (ProteomeXchange identifier PXD038165), in combination with available information from public databases, aids in optimizing the immunopeptidome characterization process. The 'Immunopeptidome Score' has been applied in a systematic analysis of protein extraction, HLA immunoprecipitation, and peptide recovery yields across several tumor cell lines enabling the selection of peptides with optimal features and, therefore, the identification of potential biomarker and therapeutic targets.

Unveiling the mechanism of cysteamine dioxygenase: A combined HPLC-MS assay and metal-substitution approach.

Mammalian cysteamine dioxygenase (ADO), a mononuclear non-heme Fe(II) enzyme with three histidine ligands, plays a key role in cysteamine catabolism and regulation of the N-degron signaling pathway. Despite its importance, the catalytic mechanism of ADO remains elusive. Here, we describe an HPLC-MS assay for characterizing thiol dioxygenase catalytic activities and a metal-substitution approach for mechanistic investigation using human ADO as a model. Two proposed mechanisms for ADO differ in oxygen activation: one involving a high-valent ferryl-oxo intermediate. We hypothesized that substituting iron with a metal that has a disfavored tendency to form high-valent states would discriminate between mechanisms. This chapter details the expression, purification, preparation, and characterization of cobalt-substituted ADO. The new HPLC-MS assay precisely measures enzymatic activity, revealing retained reactivity in the cobalt-substituted enzyme. The results obtained favor the concurrent dioxygen transfer mechanism in ADO. This combined approach provides a powerful tool for studying other non-heme iron thiol oxidizing enzymes.

Pharmacokinetic profiles of methylcobalamin in rats after multiple administration routes by a simple LC-MS/MS assay with a small volume of plasma.

Methylcobalamin (MBL) is a vitamin B12 coenzyme and is effective for treating peripheral neuropathies. Little is known about pharmacokinetics (PK) of MBL in animals, we have developed a simple assay for MBL by using only 0.01 mL of plasma for PK of MBL in rats. Under minimal light exposure (<5 lx), MBL was extracted by a simple protein precipitation using methanol and detected by liquid chromatography with tandem mass spectrometry. MBL in rat plasma at 20-10,000 ng/mL was quantified using only 0.01 mL of plasma. Relative error and relative standard deviation met the acceptance criteria in reproducibility assessments, indicating the robustness of the assay. PK of MBL was evaluated after intravenous, intramuscular, and subcutaneous administration. PK of MBL was dose proportional at 5-20 mg/kg in both intramuscular and subcutaneous administrations. Bioavailability after the two dosing routes was complete (ca. 100 %). The incurred sample reanalysis also supported that the assay is robust. The established assay was successfully applied to PK studies in rats to find that MBL showed high bioavailability after intramuscular and subcutaneous administrations.

A simplified metabolomic analysis of dried blood spots in breast cancer patients.

Breast cancer (BC) is among the most commonly diagnosed cancers. Besides mammography, breast ultrasonography and the routinely monitored protein markers, the variations of small molecular metabolites in blood may be of great diagnostic value. This study aimed to quantify specific metabolite markers with potential application in BC detection. The study enrolled 50 participants, 25 BC patients and 25 healthy controls (CTRL). Dried blood spots (DBS) were utilized as biological media and were quantified via a simplified liquid chromatography tandem mass spectrometry (LC-MS/MS) method, used in expanded newborn screening. The targeted metabolomic analysis included 12 amino acids and 32 acylcarnitines. Statistical analysis revealed a significant variation of metabolic profiles between BC patients and CTRL. Among the 44 metabolites, 18 acylcarnitines and 10 amino acids remained significant after Bonferroni correction, showing increase or decrease and enabled classification of BC patients and CTRL. The well-established LC-MS/MS protocol could provide results within few minutes. Therefore, the combination of an easy-to-handle material-DBS and LC-MS/MS protocol could facilitate BC screening/diagnosis and in the next step applied to other cancer patients, as well.

Comparative LC-MS-based metabolite profiling, antioxidant, and antibacterial properties of Bunium bulbocastanum tubers from two regions in Algeria.

Traditional herbalists have been relied on for many years by Algerians to cure a wide range of diseases. Regardless of their nutritional values, mushrooms have chemical properties that make them attractive, beneficial, and more likely to be studied by researchers, according to ethnobotanical literature on traditional phytotherapy. Among all the edible mushrooms, tubers are a type of fungus that are traditionally used in fine dining and have garnered attention recently because of their many therapeutic applications. This research delves into a meticulous analysis of bioactive constituents in Bunium bulbocastanum tubers, sourced from Mostaganem and Relizane regions, with a keen focus on polyphenols, flavonoids, and condensed tannins. The quantification of total phenolic content was executed through the Folin-Ciocalteu assay, while flavonoids were assessed via the aluminum chloride colorimetric method. In addition, condensed tannins were evaluated in this study. Antioxidant capacities were scrutinized employing the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Microbial inhibition studies were conducted against five benchmark bacterial strains, utilizing the agar disc diffusion technique. Furthermore, a comprehensive liquid chromatography-mass spectrometry (LC-MS) analysis was performed to identify and quantify bioactive compounds. The findings underscore that the Mostaganem extracts were particularly rich in polyphenols (11.65 mg GAE/g of extract) and tannins (1.30 mg CE/g of extract), while the Relizane extracts boasted significant flavonoid concentrations (9.421 mg QE/g of extract). Notably, 4-methylguaiacol (82.04 mg/L), caffeic acid dimethyl ether (27.76 mg/L), syringic acid (20.48 mg/L), and naringenin (16.05 mg/L) emerged as the predominant volatile compounds. Compositional investigation of the extracts by LC-MS confirmed the presence of various compounds that were linked to the bioactivities exhibited by B. bulbocastanum tubers. These findings demonstrate the effective antibacterial and antioxidant properties of B. bulbocastanum tubers, indicating their potential use in pharmaceutical and nutraceutical applications.

[Mechanism of liver injury induced by alcohol extract of salt-processed Psoraleae Fructus based on chemical modification of protein].

Salt-processed Psoraleae Fructus is a commonly used tonic in clinical practice. However, its usage is restricted due to the inherent toxicity. The covalent modification of proteins by reactive metabolites(RMs) plays a role in the hepatotoxicity of salt-processed Psoraleae Fructus. This study delves into the protein covalent modification by RMs generated from psoralen/isopsoralen, the primary toxic components of salt-processed Psoraleae Fructus, by liquid chromatography-mass spectrometry(LC-MS), aiming to elucidate the mechanism underlying the hepatic injury induced by salt-processed Psoraleae Fructus. Biochemical methods were utilized to measure the levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), catalase(CAT), malondialdehyde(MDA), superoxide dismutase(SOD), reduced glutathione(GSH), and glutathione S-transferase(GST) in mice. The pathological changes in the liver were observed by hematoxylin-eosin(HE) staining. Subsequently, ultra performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS) was employed to identify the primary toxic components of psoralen/isopsoralen and the RMs in salt-processed Psoraleae Fructus. Covalent bonding adducts of the toxic components/RMs with GSH and free amino acids were identified to investigate the effects of the toxic components on modification sites and patterns of amino acids. The modifications of RMs were incorporated into the variable modifications of Proteome Discoverer, and the target proteins of psoralen/isopsoralen were detected by liquid chromatography-quadrupole exactive-mass spectrometry. Lastly, Label-free quantitative proteomics was adopted to screen differential proteins, which were further subjected to KEGG and GO enrichment analyses and confirmed by qPCR. The results indicated that compared with the control group, salt-processed Psoraleae Fructus significantly elevated the ALT, AST, and MDA levels and lowered the SOD, CAT, GSH, and GST levels in a dose-dependent manner, while causing obvious vacuolization and inflammatory cell infiltration in mouse hepatocytes. Furthermore, the livers of mice in the salt-processed Psoraleae Fructus group showed the presence of five RMs of psoralen/isopsoralen, two adducts with GSH, and one adduct with cysteine. In addition, 10 proteins modified by the RMs of psoralen/isopsoralen were identified. A total of 133 differential proteins were detected in the livers of mice in the salt-processed Psoraleae Fructus group, including 92 with up-regulated expression and 41 with down-regulated expression. These differential proteins mainly involved ribosomes, rRNAs, and glutathione, affecting the proteasome pathway. The qPCR results were consistent with the differential proteins. These findings suggest that the RMs of psoralen/isopsoralen can covalently bind to GSH and modify cysteine and lysine residues of liver proteins. This covalent modification of proteins by harmful substances can potentially result in liver damage. Therefore, it can be inferred that the oxidative stress damage induced by salt-processed Psoraleae Fructus may be associated with the abnormality of proteasome and its complex, biosynthesis of ribosomes and their nucleoprotein complex, rRNA binding, and glutathione binding.

High-performance liquid chromatography-tandem mass spectrometry was used to measure 20 antidepressants in human serum.

Aim: This study used high performance liquid chromatography-tandem mass spectrometry to quantify the blood concentrations of 20 antidepressants, such as bupropion and fluoxetine, in human serum samples.Methods: After direct precipitation with a 1:9 protein precipitant of methanol and acetonitrile, serum samples were examined using high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS). The material was separated using a Poroshell 120 EC-C18 column (3.0 mm × 50 mm, 2.7 µm) and gradient elution. The mobile phases were phase A 0.01% formic acid aqueous solution (containing 2 mmol/ml ammonium acetate) and phase B methanol solution. A 0.45 ml/min flow rate was used to divide the sample and inject 5 µl. Electrospray ionization source positive ion mode and multiple reaction monitoring modes were used for analysis. Measurement was quantified using an internal standard technique.Results: Accuracy ranged from 90.3 to 114.3%, intra-day precision from 100.1 to 112.3%, inter-day precision from 100.4 to 112.6%, extraction recoveries from 85.5 to 114.5% and matrix effect from 85.6 to 98.7%.Conclusion: This approach is fast, accurate, sensitive and repeatable. It can identify 20 antidepressants in blood simultaneously. This can be used to monitor blood drug levels and medication metabolism.

[Box: see text].

Assessment of adeno-associated virus purity by capillary electrophoresis-based western.

A rigorous analytical assessment of recombinant adeno-associated virus (rAAV)-based drug products is critical for their successful development as clinical candidates. It is especially important to ascertain high purity while simultaneously ensuring low levels of impurities in the final drug product. One approach to evaluate the purity of rAAV drug products is to determine the relative stoichiometry of the three viral proteins (VPs) that comprise an rAAV capsid, and the levels of impurities in the final drug product. Here we present two capillary electrophoresis-western (CE-western) assays for quantifying (1) the relative stoichiometry of VP using the anti-AAV B1 antibody, and (2) residual levels of a baculovirus protein impurity, GP64, using the anti-GP64 antibody. In each assay, various purified samples from diverse AAV serotypes were analyzed to determine their VP ratio or GP64 levels. The ratio of VP3/VP1 in rAAV samples was correlated with biological activity, and the clearance of GP64 from the manufacturing process was demonstrated. The results obtained from both assays were further supported by liquid chromatography-mass spectrometry analyses. Overall, we report that CE-western is a high-throughput platform that utilizes low sample volumes for a rapid, sensitive, and robust assessment of the identity, composition, and purity of rAAV drug products.

Residual levels of pesticides in filet green beans from Souss-Massa, Morocco.

A total of 30 samples of filet green beans (Phaseolus vulgaris) from various popular markets in the Souss-Massa region (Morocco) were analyzed during 2021 for the presence of pesticide residues. We performed a QuEChERS extraction (Quick, Easy, Cheap, Effective, Rugged, and Safe) combined with Liquid Chromatography-Mass Spectrometry (LC-MS/MS) and Gas Chromatography-Mass Spectrometry (GC-MS/MS). The method was validated in accordance with SANTE 11312/2021 guidelines. The results of this survey indicated that 8 out of 30 green bean samples are positives (0.015 to 0.112 mg/kg). Azoxystrobin was the most frequently detected pesticide, occurring in 5 samples. For authorized pesticides, none of the samples exceeded the European Maximum residue level (EU MRL) in green beans, but residue of fipronil-unauthorized substance was detected in one sample at 0.027 mg/kg. Hence, it is important to increase inspections for locally marketed fresh green beans and align agricultural practices with regulatory requirements.

Multiple oxidative modifications on hemoglobin are elevated in breast cancer patients as measured by nanoflow liquid chromatography-tandem mass spectrometry.

Breast cancer is strongly connected with elevated oxidative stress. Oxidative modifications of hemoglobin can serve as biomarkers for monitoring oxidative stress status in vivo. The structure of hemoglobin modifications derived from malondialdehyde (MDA) in human blood hemoglobin exists as N-propenal and dihydropyridine (DHP). This study reports the simultaneous quantification of eleven modified peptides in hemoglobin derived from MDA and advanced histidine oxidation in 16 breast cancer patients and 16 healthy women using nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry. The results reveal statistically significant increases in the formation of MDA-derived N-propenal and DHP of lysine and advanced oxidation of histidine in hemoglobin of breast cancer patients with the Mann-Whitney U-test p values < 0.0001 and the AUC of ROC between 0.9277 and 1.0. Furthermore, the elevation in modified peptides is significant in patients with early stages of breast cancer. By measuring these oxidative modifications in hemoglobin from a drop of blood, the role of lipid peroxidation and oxidative stress in breast cancer can be assessed using this sensitive assay.

Absolute quantitation of human serum cystatin C: candidate reference method by 15N-labeled recombinant protein isotope dilution UPLC-MS/MS.

OBJECTIVES: Serum cystatin C (CysC) is a reliable and ideal endogenous marker for accurately assessing early changes in glomerular filtration rate (GFR), surpassing the limitations of creatinine-based estimated GFR. To improve the precision of GFR calculation, the development of strategies for accurately measuring serum CysC is crucial. METHODS: In this study, the full-length CysC pure product and fully recombinant 15N-labeled CysC internal standard were subjected to protein cleavage. Subsequently, an LC-MS/MS method was developed for the absolute quantification of serum CysC. The traceability of the method was assigned calibrator using the amino acid reference measurement procedure (RMP). It involved calibrating the instrument using an amino acid reference material with known amino acid concentrations for calibration and comparison purposes. RESULTS: The total imprecision of the method was determined to be ≤8.2 %, and a lower functional limit of quantification (LLoQ) was achieved. The recoveries ranged from 97.36 to 103.26 %. The relative bias between this candidate RMP for measurement of ERM-DA471-IFCC and the target value was 1.74 %. The linearity response was observed within the concentration range of 0.21-10.13 mg/L, with a high R2 value of 0.999. The results obtained using our method was consistent with those obtained using other certified RMPs. CONCLUSIONS: With the establishment of this highly selective and accurate serum CysC measurement method, it is now possible to assess the correlation between immunoassay results of serum CysC and the intended target when discrepancies are suspected in the clinical setting.

Metabolic profiling and spatial metabolite distribution in wild soybean (G. soja) and cultivated soybean (G. max) seeds.

Wild soybeans retain many substances significantly reduced or lost in cultivars during domestication. This study utilized LC-MS to analyze metabolites in the seed coats and embryos of wild and cultivated soybeans. 866 and 815 metabolites were identified in the seed extracts of both soybean types, with 35 and 10 significantly differing metabolites in the seed coat and embryos, respectively. The upregulated metabolites in wild soybeans are linked to plant defense, stress responses, and nitrogen cycling. MALDI-MSI results further elucidated the distribution of these differential metabolites in the cotyledons, hypocotyls, and radicles. In addition to their role in physiological processes like growth and response to environmental stimuli, the prevalent terpenoids, lipids, and flavonoids present in wild soybeans exhibit beneficial bioactivities, including anti-inflammatory, antibacterial, anticancer, and cardiovascular disease prevention properties. These findings underscore the potential of wild soybeans as a valuable resource for enhancing the nutritional and ecological adaptability of cultivated soybeans.

Phosphoinositide 3-Kinase Inhibitors from Gladiolus segetum Ker-Gawl Corms Supported by Network Pharmacology.

Gladiolus segetum Ker-Gawl corms total extract exhibited remarkable in vitro anti-proliferative effects against panel of cancer cell lines; including human colon carcinoma (Caco-2), human breast cancer (MCF7) and hepatocellular carcinoma (HepG2) cell lines with IC50 values of 7.4, 9.1 and 11.2 µg/ml, respectively. The total ethanolic extract of G. segetum Ker-Gawl corms was subjected to untargeted metabolomics profiling using LC-HR-ESI-MS, which revealed the presence of various clusters of phytoconstituents as triterpenes, anthraquinones, flavonoids and phenolic derivatives. Network pharmacology study was performed for all identified compounds, the formed networks identified 73 intersected genes. The diagrammatic illustration of the top pathways revealed that phosphoinositide 3-kinase (PI3K) gene is the effective dominant gene in the top four KEGG pathways. Upon molecular docking and molecular dynamics investigation, kaempferol-3-O-glucopyranoside was suggested to be key anticancer metabolite. Interestingly, cytotoxic investigation of this compound revealed potential activity against the tested cancer cell lines (Caco-2, MCF7 and HepG2) with IC50 values of 6.2, 8.5 and 9.3 µg/ml, respectively. The present study highlighted the potential of G. segetum Ker-Gawl as a promising source of interesting anticancer scaffolds.

Poweromin X Ten, a polyherbal formulation improves male sexual function: In vivo and network pharmacology study.

Introduction: Poweromin X Ten (PXT) is a polyherbal formulation, traditionally used to enhance male sexual function. However, the safety and benefits of PXT have not been scientifically evaluated. Therefore, the present study investigated the toxicity and aphrodisiac potential of PXT in male rats and explored its principal mechanisms of action. Methods: Male Wistar rats were orally administered PXT (50 or 100 mg/kg) for 28 days, and sexual activity parameters, including latency and frequency of mounting and intromissions, were studied. The reproductive toxicity and spermatogenic potential were also examined. Furthermore, dopamine and serotonin levels in brain regions associated with sexual activity were assessed. Network analysis was used to identify the key bioactive compounds and their core targets involved in their beneficial actions. Results: Treatment with PXT improved sexual activity in male rats, as evidenced by reduced mounting and intromission latency and a significant increase in mount frequency. Moreover, PXT exhibited spermatogenic potential and did not induce reproductive toxicity. Notably, treatment with 50 mg/kg PXT elevated dopamine levels in median preoptic area and hypothalamus. Pathway analysis indicated that PXT primarily modulated the PI3K-Akt, calcium, and MAPK signalling pathways to enhance male sexual function. Network analysis identified macelignan, ß-estradiol, testosterone, and paniculatine as key bioactive components of PXT, which likely act through core targets, such as androgen receptor (AR), Mitogen-activated protein kinase 3 (MAPK3), epidermal growth factor receptor (EGFR), estrogen receptor 1 (ESR1), and vascular endothelial growth factor (VEGF) to facilitate the improvement of male sexual function. Conclusion: Study results suggest that PXT is a safer alternative with aphrodisiac and spermatogenic potential. These effects are partly attributed to the enhanced dopamine levels in the brain. Furthermore, this study provides insights into the specific signalling pathways and bioactive compounds that underlie the improvements in male sexual function associated with PXT.

LC-ESI-MS/MS method validation for simultaneous quantification of FDA-approved anticancer agents futibatinib and binimetinib in rat plasma: Insights from preclinical pharmacokinetics.

This study investigates the combination of FGFR inhibitor futibatinib (FTB) and MEK inhibitor binimetinib (BTB) for KRASmt NSCLC therapy. An analytical method was developed and validated for measuring FTB and BTB concentrations in rat plasma, adhering to USFDA guidelines. Using liquid-liquid extraction on 45-µL plasma samples, a 6.5-min run time was achieved. The linear calibration curve ranged from 2 to 100 ng/mL. Intra-day and inter-day accuracy ranged between 92.06% and 100.08%. Four blank injections post high-concentration samples resolved significant carryover. Extraction recoveries averaged 92.06% to 102.37% across concentrations. No significant endogenous interference was detected in blank plasma. The LLOQ for both drugs was 2.0 ng/mL. Selectivity, matrix effects, stability, and dilution integrity met the acceptance criteria. The method assessed FTB and BTB interaction potential in combination therapy at 5 mg/kg. The findings provide essential pharmacokinetics insights for future clinical trials.