High-performance Liquid Chromatography-electrospray Ionization-tandem Mass Research Articles

Improvement of Alginate Extraction from Brown Seaweed (Laminaria digitata L.) and Valorization of Its Remaining Ethanolic Fraction.

This study aimed to improve the conventional procedure of alginate isolation from the brown seaweed (Laminaria digitata L.) biomass and investigate the possibility of further valorization of the ethanolic fraction representing the byproduct after the degreasing and depigmentation of biomass. The acid treatment of biomass supported by ultrasound was modeled and optimized regarding the alginate yield using a response surface methodology based on the Box-Behnken design. A treatment time of 30 min, a liquid-to-solid ratio of 30 mL/g, and a treatment temperature of 47 °C were proposed as optimal conditions under which the alginate yield related to the mass of dry biomass was 30.9%. The use of ultrasonic radiation significantly reduced the time required for the acid treatment of biomass by about 4 to 24 times compared to other available conventional procedures. The isolated alginate had an M/G ratio of 1.08, which indicates a greater presence of M-blocks in its structure and the possibility of forming a soft and elastic hydrogel with its use. The chemical composition of the ethanolic fraction including total antioxidant content (293 mg gallic acid equivalent/g dry weight), total flavonoid content (14.9 mg rutin equivalent/g dry weight), contents of macroelements (the highest content of sodium, 106.59 mg/g dry weight), and microelement content (the highest content of boron, 198.84 mg/g dry weight) was determined, and the identification of bioactive compounds was carried out. The results of ultra high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis confirmed the presence of 48 compounds, of which 41 compounds were identified as sugar alcohol, phenolic compounds, and lipids. According to the 2,2-diphenyl-1-picrylhydrazyl assay, the radical scavenging activity of the ethanolic fraction (the half-maximal inhibitory concentration of 42.84 ± 0.81 μg/mL) indicated its strong activity, which was almost the same as in the case of the positive control, synthetic antioxidant butylhydroxytoluene (the half-maximal inhibitory concentration of 36.61 ± 0.79 μg/mL). Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus) were more sensitive to the ethanolic fraction compared to Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Shigella sonnei). The obtained results indicated the possibility of the further use of the ethanolic fraction as a fertilizer for plant growth in different species and antifouling agents, applicable in aquaculture.

Unveiling Pharmacokinetics and Drug Interaction of Linagliptin and Pioglitazone HCl in Rat Plasma Using LC-MS/MS.

The linagliptin (LIN) and pioglitazone HCl (PIO) combination, currently undergoing phase III clinical trials for diabetes mellitus treatment, demonstrated significant improvements in glycemic control. However, the absence of an analytical method for simultaneous determination in biological fluids highlights a crucial gap. This underscores the pressing need for sensitive bioanalytical methods, emphasizing the paramount importance of developing such tools to advance diabetes management strategies and enhance patient care. Herein, a sensitive reverse-phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for simultaneous determination of LIN and PIO in rat plasma using alogliptin as an internal standard. Chromatographic separation was performed on an Agilent Eclipse Plus C18 (4.6 × 100 mm, 3.5 μm) using an isocratic mobile phase system consisting of ammonium formate (pH 4.5) and methanol using an acetonitrile-induced protein precipitation technique for sample preparation. Multiple reaction monitoring in positive ion mode was used for quantitation of the precursor to production at m/z 473.2 → 419.9 for LIN, 357.1 → 134.2 for PIO, and 340.3 → 116.1 for ALO. The linearity range was 0.5 to 100 and 1 to 2000 ng/mL for LIN and PIO, respectively. The developed method was validated as per US-FDA guidelines and successfully applied to clinical pharmacokinetic and drug-drug interaction studies with a single oral administration of LIN and PIO in rat plasma. Pharmacokinetic parameters of LIN were significantly influenced by the concomitant administration of PIO and vice versa. Molecular modeling revealed the significant interaction of LIN and PIO with P-glycoprotein. Therefore, the drug-drug interaction between LIN and PIO deserves further study to improve drug therapy and prevent dangerous adverse effects.

Chemical exploration of different extracts from Phytolacca americana leaves and their potential utilization for global health problems: ın silico and network pharmacology validation

Phytolacca americana L. is of great interest as a traditional additive in various folk remedies in several countries, including Turkey. We aimed to determine the chemical profile (assisted by high-Performance liquid chromatography-electrospray ionization-tandem mass apectrometry (HPLC-ESI-MS/MS) experiments of three extracts obtained by different polarity solvents viz. ethyl acetate (to extract semipolar compounds), methanol and water (to extract highly polar metabolites) from P. americana leaves. Their anti-diabetic effects were investigated in vitro by assessing their inhibition toα-amylase and α-glucosidase. Assessment of the neuroprotective potential of the three extracts was carried out against acetyl-(AChE) and butyryl-(BChE) cholinesterase enzymes. HPLC-ESI-MS/MS experiments showed a total of 17 chromatographic peaks primarily classified to six flavonoids, two saponins, and six fatty acids. Antioxidant assays revealed remarkable activity for the ethyl acetate and methanol extracts. The BChE inhibition was considerably more significant (4.08 mg galantamine equivalent (GALAE)/g) for the ethyl acetate extract, whereas the methanol extract had good inhibitory efficacy for AChE (2.05 mg GALAE/g). Through network pharmacology, the compounds’ mechanism of action of targeted key gene in their associated diseases were identified. The hubb gene signal transducer and activator of transcription 3 (STAT3) and tumour necrosis factor (TNFα) where the P. americana compound’s site of action in inflammation bowel disease. The results offer possibilities for the prospective application of P. americana in metabolic regulation, blood glucose control, and as a source of bioactive compounds with cholinesterase enzyme inhibitory characteristics which could be of relevance in the cosmetic or pharmaceutical industry for combating melanogenesis. Communicated by Ramaswamy H. Sarma

Evaluation of disposable protective garments against epoxy resin permeation and penetration from anti-corrosion coatings.

Epoxy-based resin formulations are a frequent cause of allergic and irritant contact dermatitis in the construction and painting industries. Cases of epoxy resin contact dermatitis continue to persist across many sectors and are likely attributable to the growing use of epoxy products, including epoxy-based anti-corrosion coatings and inadequate skin protection. There are no published performance data against epoxy resins for garment materials and gloves to guide proper material selection in the workplace. The objective of this study was to evaluate the resistance of 5 protective garment materials against permeation and penetration by bisphenol A diglycidyl ether and its higher oligomers found commonly in epoxy-based anti-corrosion coatings. Five disposable garment materials were evaluated for resistance to bisphenol A diglycidyl ether monomers and oligomers during contact with epoxy-based anti-corrosion coatings, including latex gloves, nitrile gloves, Tyvek coveralls, polypropylene/polyethylene (PP/PE) coveralls, and a cotton T-shirt. A permeation test cell system was used to evaluate each garment material against an epoxy-based zinc-rich primer and an epoxy-based intermediate coating using a realistic application method. Glass fiber filters were used to collect permeating and penetrating epoxy resin during a 120-min test period. Bisphenol A diglycidyl ether quantification was performed with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Paint loading, coating thickness, and homogeneity were assessed on polytetrafluoroethylene filters sprayed in series in permeation test cells. Latex gloves provided the least resistance to permeation by BADGE in coating formulations, with a maximum cumulative permeation over the 2-h test interval of 21.7 ng cm-2 with the primer and 513.8 ng cm-2 with the intermediate coating product. Nitrile gloves were not permeated by either coating formulation. The Tyvek coveralls provided greater protection as compared to the PP/PE coveralls. The cotton T-shirt was penetrated by bisphenol A diglycidyl ether more frequently than any of the tested garment materials and resulted in a maximum cumulative penetration of 128 ng cm-2 with the primer and 28.0 ng cm-2 with the intermediate coating. Although all the garment materials evaluated during this study provided sufficient protection to prevent cumulative permeation in excess of the established acceptable permeation thresholds, the use of nitrile gloves and Tyvek coverall is highly recommended to minimize skin exposure to bisphenol A diglycidyl ether. We recommend cotton T-shirts to be used under Tyvek coveralls as a secondary layer of skin protection and for added comfort, but not as a primary protection layer.

Associations of urinary caffeine and caffeine metabolites with metabolic syndrome in US adults.

The relationship between caffeine and metabolic syndrome (MetS) has only been evaluated from the perspective of caffeine consumption. The association between urinary caffeine and MetS is still unclear. This study examined the associations between urinary caffeine and its metabolites and MetS and its components among adults. Data from the United States (US) National Health and Nutrition Examination Survey (NHANES) 2011-2014 was analyzed. NHANES is a stratified, multi-stage survey of all non-institutionalized persons in the US. A total of 2,394 subjects aged ≥ 18 years without missing data were selected in this study. Urinary caffeine and caffeine metabolite levels were quantified using high-performance liquid chromatography-electrospray ionization-tandem quadrupole mass spectrometry (HPLC-ESI-MS/MS) with stable isotope-labeled internal standards. We performed principal components analysis (PCA) to investigate the underlying correlation structure of 15 features of urinary caffeine and its metabolites and then used these principal components (PCs) as independent variables to conduct logistic regression analysis with or without restricted cubic spline (RCS) terms to explore the associations between caffeine metabolites and MetS. Two main PCs that were derived from the PCA explained 90.67% of the total variance of caffeine and its metabolites. The first PC (PC1, strongly correlated with 1-MU, 1,3-DMU, 1,7-DMU, 1,3,7-TMU, 1-MX, 1,3-DMX, 1,7-DMX, 1,3,7-TMX, and AAMU) was positively correlated with risk of MetS (OR = 1.27, p < 0.001) and all its components (all ORs > 1, all p-values < 0.001) in the unadjusted models, while in the adjusted models, it was positively correlated with MetS (OR = 1.16, p = 0.042) and central obesity (OR = 1.22, p < 0.001). In the unadjusted model, there were significant associations between the second PC (PC2, correlated with 3-MU, 7-MU, 3,7-DMU, 3-MX, 7-MX, and 3,7-DMX) and MetS (OR = 1.11, P = 0.030) and central obesity (OR = 1.16, P < 0.001), while in the adjusted models (adjustment variables include gender, age, race/ethnicity, education level and income-poverty ratio, smoking status, drinking, and physical activity), PC2 was positively associated with MetS (OR = 1.15, p = 0.035) and central obesity (OR = 1.15, p = 0.005) and negatively associated with raised triglycerides (TG) (OR = 0.84, p = 0.008). Moreover, we observed U-shaped associations between PC1 and the risk of raised TG both in unadjusted (Pnon-linear = 0.017) and adjusted (Pnon-linear = 0.014) models. Urinary caffeine metabolites were positively associated with the risk of MetS and its components through different linear or non-linear patterns.

Simultaneous quantification of seven B vitamins in human faeces by stable isotope label-based high-performance liquid chromatography-tandem mass spectrometry

B vitamins in the human distal gut are primarily derived from the gut microbiota because daily dietary vitamins are fully absorbed in the small intestine under normal dietary and physiological conditions. Quantitative determination of B vitamins in the distal gut and faecal samples is crucial for understanding the intricate relationship between gut B vitamins, gut microbiota, and host health. In this study, we developed a rapid, robust, and reliable method with a simple extraction procedure for the simultaneous analysis of seven B vitamins in human faeces using high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (HPLC-ESI-MS/MS) with stable isotope-labelled internal standards. A protein precipitation approach using methanol as the precipitant was employed to extract vitamin B from human faecal samples. Seven B vitamins were adequately separated and quantified within 9 min by HPLC-ESI-MS/MS with a Pursuit PFP column (2.0 ×150 mm, 3.0 µm), including vitamins B1, B2, B3, B5, pyridoxic acid, pyridoxine, and B7. The lower limits of quantification were within the range of 0.1–25 ng mL−1. The intra-day and inter-day precision and accuracy were both within 15 %. The validated method was successfully applied to 55 faecal samples collected from healthy individuals, patients with type 2 diabetes, and obese patients. Compared with healthy controls, obese patients had lower faecal concentrations of vitamins B1 and B3 and pyridoxic acid, whereas patients with type 2 diabetes had lower faecal concentrations of vitamins B1 and B5.

The associations of coffee consumption, coffee types, and caffeine metabolites with periodontitis: Results from NHANES 2009-2014.

Coffee is one of the world's most popular beverages and is the main dietary source of caffeine for most people. The various molecular effects of caffeine suggest that it may enhance bone loss. The purpose of the present study was to investigate the relationship of coffee, coffee types, and caffeine metabolites with periodontitis. Data were obtained from the National Health and Nutrition Examination Survey (NHANES) 2009-2014. Total coffee and different types of coffee consumption were acquired through a 24-h dietary recall. Urinary caffeine metabolites were quantified using high-performance liquid chromatography-electrospray ionization-tandem quadrupole mass spectrometry (HPLC‒ESI‒MS/MS). The association of coffee, coffee types, and caffeine metabolites with periodontitis and its severity were assessed using multivariable logistic regression. A total of 3309 eligible participants were included. After adjusting for potential confounding variables, a positive association was observed between coffee consumption (particularly certain types of coffee) and periodontitis. Notably, a positive correlation was also found between total coffee intake and the severity of periodontitis. Additionally, for urinary caffeine metabolites, there was a significant positive association between 1-methyluric acid (1-MU), 1,3-dimethyluric acid (1,3-DMU), 3,7-dimethyluric acid (3,7-DMU), 1,7-dimethylxanthine (1,7-DMX), or 5-actlyamino-6-amino-3-methyluracil (AAMU) and periodontitis, with adjusted odds ratios and 95% confidence intervals of 1.10 (1.02, 1.19), 1.86 (1.05, 3.29), 0.94 (0.90, 0.98), 1.29 (1.03, 1.62), and 1.15 (1.05, 1.26), respectively. The present study suggests a positive association of coffee intake (especially certain coffee types) and caffeine metabolites (1-MU, 1,3-DMU, 3,7-DMU, 1,7-DMX, and AAMU) with periodontitis and its severity.

Profiles, antioxidative and ACE inhibitory activity of peptides released from fermented buttermilk before and after simulated gastrointestinal digestion

Bioactive peptides, released from buttermilk by fermentation and/or gastrointestinal proteases, may have health promoting effects. Thus, a comprehensive analysis of the peptide fraction of fermented buttermilk, before and after different phases of simulated gastrointestinal digestion, was performed using ultra high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS). Results showed that digestion simulation substantially changed the peptide profile of fermented buttermilk. A total of 81, 120 and 46 peptides were identified in fermented buttermilk, its gastric and intestinal digests, respectively. These peptides released mostly from β-casein followed by αs1-casein, κ-casein and β-lactoglobulin. In addition, 14 peptides released from milk fat globule membrane proteins (lactadherin, butyrophilin and GlyCAM-1). Bioactivity, mainly angiotensin converting enzyme (ACE) inhibitory activity, has been reported before for only 54 of the detected peptides. Radical scavenging, ferric reducing and ACE inhibitory activities of fermented buttermilk peptides increased significantly after digestion, indicating promotion in fermented buttermilk-peptide bioactivity by gastrointestinal digestion.

OCCURRENCE OF METFORMIN IN ENVIRONMENTAL WATER SAMPLES AND COMPARISON WITH CONSUMPTION DATA FROM A SURROUNDING HOSPITAL OVER 5 YEARS: A RETROSPECTIVE CASE STUDY.

The environmental occurrence of metformin has been frequently world-widely reported. Despite the diabetes susceptibility in the Chinese population, the studies on occurrence of metformin as environment disruptor in China are insufficient. To determine the occurrence trends and possible environmental pollution sources of metformin as an emerging micropollutant. High-performance liquid chromatography-electrospray ionization-tandem mass spectrometry system was used to detect the metformin levels in water samples collected from the Xi'an city Moat, China once a year from 2017 to 2021. Correlations among the metformin levels in moat water, in surrounding hospital wastewater, and hospital metformin consumption data were assessed using Pearson, Spearman and Kendall's tau-b correlation coefficients. Occurrence of metformin was found in Xi'an city Moat water with levels in the range of 304-793 ng/L. Significant correlations were found between the metformin levels in city moat water and the total (or outpatient) metformin utilization data of the hospital. Data suggested the potential environmental issues posed by metformin in Xi'an city in China. The metformin consumption volume in the surrounding hospitals, especially at the outpatient services, could be used to predict the metformin concentrations in the moat water.

Urine caffeine metabolites are positively associated with cognitive performance in older adults: An analysis of US National Health and Nutrition Examination Survey (NHANES) 2011 to 2014

The aim of this study was to explore urine caffeine metabolites in relation to cognitive performance among 2011-2014 National Health and Nutrition Examination Survey participants aged ≥60 years. We hypothesized that urine caffeine metabolites were positively associated with cognition in older adults. Caffeine and 14 of its metabolites were quantified in urine by use of high-performance liquid chromatography-electrospray ionization-tandem quadruple mass spectrometry with stable isotope labeled internal standards. Cognitive assessment was based on scores from the word learning and recall modules. Participants were categorized based on the quartiles of caffeine and its metabolites level. The association between caffeine metabolites and each cognitive dimension was analyzed using multiple logistic regression analysis in adjusted models. Stratification analyses by gender were also performed. For CERAD test, there was a significant association between 1-methyluric acid (OR=0.62, 95% CI: 0.42 to 0.92), 7-methylxanthine(OR=0.49, 95% CI: 0.27 to 0.89), theophylline (OR=0.52, 95% CI: 0.29 to 0.92), as well as paraxanthine (OR=0.49, 95% CI: 0.27 to 0.88) and cognitive function. For animal fluency test, there was a positive association between theophylline (TP) (OR=0.44, 95% CI: 0.22 to 0.89) and cognitive function. The trend that the risk of low cognitive function decreased with increasing concentration of 1-methylxanthine (P trend=0.0229) was also observed. Furthermore, the same trend existed for 3-methylxanthine (p trend = 0.0375) in men. In conclusion, there was a significant positive association between urine caffeine metabolites and cognitive performance in older adults, particularly for theophylline, paraxanthine and caffeine; and the association might be dependent on gender.

Determination and enantioselective separation of zilpaterol in human urine after mimicking consumption of contaminated meat using high-performance liquid chromatography with tandem mass spectrometry techniques.

The synthetic β-adrenoreceptor agonist zilpaterol is legitimately used as an animal feed supplement in selected countries due to its known effects on lipolysis and protein biosynthesis. These pharmacological characteristics of zilpaterol have contributed to its classification as doping agent in sport by the World Anti-Doping Agency. However, the use as a feed supplement can lead to residues of the drug in edible tissues and, possibly, also in the urine of consumers. To provide urinary elimination profiles of microdosed zilpaterol and to determine whether the ingestion of zilpaterol below or at the acceptable daily intake level of 0.04 μg/kg bodyweight can result in an adverse analytical finding (AAF) in doping controls, healthy volunteers were administered single or multiple oral doses of 0.5μg or 3μg zilpaterol to mimic ingestion of contaminated cattle meat. Urine samples were collected and analyzed using a validated high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method and a newly developed chiral high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method. Urinary peak concentrations of zilpaterol were observed for all volunteers 1.5-12.5h after ingestion, and maximum levels >5ng/mL, which would constitute an AAF in doping controls, were found after the intake of 3μg of zilpaterol on five consecutive days in one out of five study participants. Noteworthy, the enantiomeric ratio of excreted zilpaterol remained constant over time. This study provides first insights into the urinary excretion of microdosed zilpaterol. Furthermore, a method was successfully developed and applied for the separation of the zilpaterol enantiomers with mass spectrometric detection.

Effects of gibberellin applications before flowering on the phenotype, ripening, and flavonoid compounds of Syrah grape berries.

Vitis vinifera L. cv. Syrah grapevines in most Chinese viticulture regions generally have compact clusters that increase the susceptibility to diseases and inhibit coloration of the inner berries. Gibberellic acid (GA3 ) is a plant growth regulator that is widely used during grape cultivation to elongate the rachis, control fruit set, and decrease cluster compactness. In this study, Syrah grapevines were treated with GA3 before flowering in 2019 and 2020 to determine the optimal GA3 treatment concentrations and times for decreasing bunch compactness, while minimizing the negative effects on the wine grape cluster weight. Pre-flowering GA3 applications at 3, 5, and 7mg L-1 , especially treatment at 20 days before flowering, decreased Syrah grape bunch compactness by decreasing the fruit set rate and promoting bunch elongation, with minimal adverse effects on the healthy grape cluster weight in both years. The 7mg L-1 GA3 treatment at 20 days before flowering significantly increased reducing sugar, total phenolic, tannin, and total anthocyanin contents of Syrah grape berries in 2019 and 2020. Moreover, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, hierarchical cluster, and principal component analysis results indicated GA3 applications before flowering (3, 5, and 7mg L-1 ) significantly affected the accumulation of different anthocyanins in Syrah grape berries. Notably, the application of 7mg L-1 GA3 at 20 days before flowering resulted in the highest anthocyanin content. Pre-flowering gibberellin application can decrease bunch compactness and improve the quality of Syrah grape berries. These findings reflect the potential utility of gibberellin treatments for decreasing cluster compactness and increasing the quality of wine grapes. © 2022 Society of Chemical Industry.

Hop Tannins as Multifunctional Tyrosinase Inhibitor: Structure Characterization, Inhibition Activity, and Mechanism.

The application of hops could be extended to obtain higher commercial values. Tannins from hops were assessed for their tyrosinase inhibition ability, and the associated mechanisms were explored. Nuclear magnetic resonance (NMR) and high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MS/MS) revealed that the hop tannins were characterized as condensed tannins with (epi)catechin and (epi)gallocatechin as subunits and an average polymerization degree of 10.32. Tyrosinase inhibition assay indicated that hop tannins had an IC50 = 76.52 ± 6.56 μM. Kinetic studies of the inhibition processes indicated the tannins provided inhibition through competitive–uncompetitive mixed reactions. In silico molecule docking showed that tannins were bound to the active site of tyrosinase via hydrogen and electrovalent bonds. Circular dichroism (CD) observed the structural variation in the tyrosinase after reacting with the tannins. Fluorescence quenching analysis and free radical scavenging assays indicated that the tannins had copper ion chelating and antioxidant activities, which may also contribute to inhibition. The intracellular inhibition assay revealed that the melanin was reduced by 34.50% in B16F10 cells. These results indicate that these tannins can be applied as whitening agents in the cosmetics industry.

Cytotoxicity of Callerya speciosa Fractions against Myeloma and Lymphoma Cell Lines.

Callerya speciosa is widely distributed in tropical and subtropical countries and is traditionally used for preventing numerous disorders. In this study, a bioguided fractionation of ethyl acetate extract (SE) from C. speciosa root was carried out to target antioxidant and cytotoxic activities. Of the four fractions (SE1-SE4) obtained by column chromatography, SE4 had the strongest anti-radical ability in the DPPH and ABTS assays (IC50 = 0.05 and 0.17 mg/mL, respectively), with results close to butylated hydroxytoluene (BHT), a common antioxidant agent. The cytotoxic activities against the selected cells were analyzed in this study by MTT assay. Accordingly, SE2, SE3, and SE4 significantly inhibited the viability of multiple myeloma cell lines, comprising U266 (IC50 = 0.38, 0.09, and 0.11 mg/mL, respectively) and KMS11 (IC50 = 0.09, 0.17, and 0.15 mg/mL, respectively), mantle cell lymphoma Mino (IC50 = 0.08, 0.16, and 0.15 mg/mL, respectively), and the noncancerous cell line LCL (IC50 = 0.40, 0.32, and 0.21 mg/mL, respectively). At a concentration of 125 µg/mL, SE2, SE3, and SE4 induced the cell apoptosis of U266 (32.2%, 53.2%, and 55.6%, respectively), KMS11 (36.9%, 40.8%, and 47.9%, respectively), Mino (36.6%, 39.8%, and 22.0%, respectively), and LCL (12.4%, 17.5%, and 23.5%, respectively) via annexin V assay. The dominant compounds detected in fractions by high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC-ESI-MS/MS), were identified as isoflavones. This is the first report describing C. speciosa as a promising natural source of antileukemia and antimyeloma agents, which may be useful for the development of blood cancer treatments.

Comparative Evaluation of the Phytochemical Profiles and Antioxidant Potentials of Olive Leaves from 32 Cultivars Grown in China.

Olives (Olea europaea L.) are a significant part of the agroindustry in China. Olive leaves, the most abundant by-products of the olive and olive oil industry, contain bioactive compounds that are beneficial to human health. The purpose of this study was to evaluate the phytochemical profiles and antioxidant capacities of olive leaves from 32 cultivars grown in China. A total of 32 phytochemical compounds were identified using high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry, including 17 flavonoids, five iridoids, two hydroxycinnamic acids, six triterpenic acids, one simple phenol, and one coumarin. Specifically, olive leaves were found to be excellent sources of flavonoids (4.92–18.29 mg/g dw), iridoids (5.75–33.73 mg/g dw), and triterpenic acids (15.72–35.75 mg/g dw), and considerable variations in phytochemical content were detected among the different cultivars. All tested cultivars were classified into three categories according to their oil contents for further comparative phytochemicals assessment. Principal component analysis indicated that the investigated olive cultivars could be distinguished based upon their phytochemical profiles and antioxidant capacities. The olive leaves obtained from the low-oil-content (<16%) cultivars exhibited higher levels of glycosylated flavonoids and iridoids, while those obtained from high-oil-content (>20%) cultivars contained mainly triterpenic acids in their compositions. Correspondingly, the low-oil-content cultivars (OL3, Frantoio selection and OL14, Huaou 5) exhibited the highest ABTS antioxidant activities (758.01 ± 16.54 and 710.64 ± 14.58 mg TE/g dw, respectively), and OL9 (Olea europaea subsp. Cuspidata isolate Yunnan) and OL3 exhibited the highest ferric reducing/antioxidant power assay values (1228.29 ± 23.95 mg TE/g dw and 1099.99 ± 14.30 mg TE/g dw, respectively). The results from this study may be beneficial to the comprehensive evaluation and utilization of bioactive compounds in olive leaves.

Counter-current fractionation-assisted and bioassay-guided separation of active compounds from cranberry and their interaction with α-glucosidase

An effective method for the selection of active compounds from cranberry based on counter-current fractionation and bioassay-guided separation was established. Cranberry extract showed potential α-glucosidase inhibitory activity. The active compounds were extracted by different solvents and enriched in water. The water extract was divided into eight fractions via high-speed counter-current chromatography to further track the active compounds. Results indicated that the α-glucosidase inhibitory activity of F7 was remarkable higher than the other fractions. Four compounds, including delphinidin-3-glucoside (D3G), cyanidin-3-glucoside, cyanidin-3-rutinoside, and pelargonidin-3-glucoside, were separated from F7 through column chromatography, and their structures were identified by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, 1H nuclear magnetic resonance (NMR), and 13C NMR. D3G showed the strongest α-glucosidase inhibitory activity among the compounds. Moreover, the interaction mechanism between α-glucosidase and D3G was clearly characterized and described by spectroscopic analyses and molecular docking. D3G could spontaneously bind with α-glucosidase to form complexes through hydrophobic interaction. The secondary structure of α-glucosidase changed in varying degrees after complexation with D3G. The α-helix and β-turn contents of α-glucosidase markedly decreased, whereas the irregular coil increased. These findings provide a new insight into the interaction between α-glucosidase and D3G.

Counter-Current Fractionation-Assisted Bioassay-Guided Separation of Active Compound from Blueberry and the Interaction between the Active Compound and α-Glucosidase

An efficient strategy for the selection of active compounds from blueberry based on counter-current fractionation and bioassay-guided separation was established in this study. Blueberry extract showed potential α-glucosidase inhibitory activity. After extraction by different solvents, the active components were enriched in water. The water extract was divided into six fractions via high-speed counter-current chromatography to further track the active components. Results indicated that the α-glucosidase inhibition rate of F4 was remarkable higher than the others. Cyanidin-3-glucoside (C3G) with a purity of 94.16% was successfully separated from F4 through column chromatography, and its structure was identified by ultraviolet spectral, Fourier-transformed infrared spectroscopy, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, 1H nuclear magnetic resonance (NMR), and 13C NMR. The interaction mechanism between C3G and α-glucosidase was clearly characterized and described by spectroscopic methods, including fluorescence and circular dichroism (CD) in combination with molecular docking techniques. C3G could spontaneously bind with α-glucosidase to form complexes by hydrogen bonds. The secondary structure of α-glucosidase changed in varying degrees after complexation with C3G. The α-helical and β-turn contents of α-glucosidase decreased, whereas the β-sheet content and the irregular coil structures increased. Molecular docking speculated that C3G could form hydrogen bonds with α-glucosidase by binding to the active sit (Leu 313, Ser 157, Tyr 158, Phe 314, Arg 315, and two Asp 307). These findings may be useful for the development of functional foods to tackle type 2 diabetes.

Regulatory effects of hawthorn polyphenols on hyperglycemic, inflammatory, insulin resistance responses, and alleviation of aortic injury in type 2 diabetic rats

Hawthorn polyphenol extract (HPE) is beneficial for patients with type 2 diabetes (T2D). However, the mechanism underlying its beneficial effects remains unclear. We investigated the inhibitory effects and mechanisms of HPE on insulin resistance, inflammation, and aortic injury in T2D rats, using metformin (MF) as a positive control. High-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) was used to determine the primary polyphenols in HPE. Hematoxylin & Eosin (H&E) staining was used to evaluate pathological conditions of the skeletal muscle, liver, and aorta vessels in each group. The levels of serum and intestinal tissue oxidative stress, tumor necrosis factor α (TNF-α), and inflammatory interleukin-6 (IL-6) were also assessed. Western blotting was used to evaluate protein expression levels in the associated molecular pathway. Volatile organic compounds (VOCs) from colon contents were determined using headspace-gas chromatography-ion mobility chromatography. Our results showed that supplementation with 300 mg HPE/kg body weight over four weeks significantly improved total cholesterol (TC), total triglyceride (TG), insulin, and lipopolysaccharide (LPS) levels in diabetic rats (p < 0.01). The lesions of skeletal muscle, liver, and aorta in diabetic rats were significantly improved. HPE supplementation also significantly downregulated the inflammatory factors (IL-6, TNF-α, and MCP-1) in the liver of diabetic rats via the SIRT1/AMPK/NF-κB signaling pathway. Furthermore, HPE significantly reduced insulin resistance in T2D rats by upregulating the phosphorylation of glucose absorption protein (GLUT4) and insulin resistance-associated proteins, p-IRS1, p-AKT, and p-PI3K, in the rat liver (p < 0.01). The findings show that HPE could also alleviate aortic injury by activating SIRT1 and regulating the NF-κB and Wnt2/β-catenin signaling pathways. Overall, the results of this study suggest that both HPE and MF have similar inhibitory effects on T2D in rats and that HPE could be used as a functional food component in the adjuvant treatment of T2D.

In Vivo Efficacy and Metabolism of the Antimalarial Cycleanine and Improved In Vitro Antiplasmodial Activity of Semisynthetic Analogues.

Bisbenzylisoquinoline (BBIQ) alkaloids are a diverse group of natural products that demonstrate a range of biological activities. In this study, the in vitro antiplasmodial activity of three BBIQ alkaloids (cycleanine [compound 1], isochondodendrine [compound 2], and 2'-norcocsuline [compound 3]) isolated from the Triclisia subcordata Oliv. medicinal plant traditionally used for the treatment of malaria in Nigeria are studied alongside two semisynthetic analogues (compounds 4 and 5) of cycleanine. The antiproliferative effects against a chloroquine-resistant Plasmodium falciparum strain were determined using a SYBR green 1 fluorescence assay. The in vivo antimalarial activity of cycleanine is then investigated in suppressive, prophylactic, and curative murine malaria models after infection with a chloroquine-sensitive Plasmodium berghei strain. BBIQ alkaloids (compounds 1 to 5) exerted in vitro antiplasmodial activities with 50% inhibitory concentration (IC50) at low micromolar concentrations and the two semisynthetic cycleanine analogues showed an improved potency and selectivity compared to those of cycleanine. At oral doses of 25 and 50 mg/kg body weight of infected mice, cycleanine suppressed the levels of parasitemia and increased mean survival times significantly compared to those of the control groups. The metabolites and metabolic pathways of cycleanine were also studied using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Twelve novel metabolites were detected in rats after intragastric administration of cycleanine. The metabolic pathways of cycleanine were demonstrated to involve hydroxylation, dehydrogenation, and demethylation. Overall, these in vitro and in vivo results provide a basis for the future evaluation of cycleanine and its analogues as leads for further development.

Development and validation of a UHPLC-ESI-MS/MS method for the quantification of artepillin C in Brazilian green propolis

Objectives: The aim of this study was the development and validation of a fast method to quantify artepillin C in green propolis using ultra high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS). Methods: High purity (97.8%) artepillin C was isolated from green propolis using chromatography techniques. Quantification was performed using a C18(2.1 x 100 mm; 1.7 μm) column, gradient of water and methanol (with 0.01% formic acid) as mobile phase, at a flow rate of 0.4 mL/min and 45 ºC in temperature. A mass spectrometer operated in selected reaction monitoring mode to monitor the deprotonated molecular ion of artepillin C (m/z 299) &gt; fragment ion (m/z 200.12). Several parameters such as specificity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, accuracy, and robustness were determined. Results: The method was linear in the 50 – 400 μg/mL range (r2 = 0.9906), showing LOD = 10.79 μg/mL and LOQ = 32.70 μg/mL with satisfactory intra-day and inter-day precision with relative standard deviation (RSD %) of 1.9% and 3.4%, respectively. The accuracy showed recovery of 93-104%, the method was robust and artepillin C was quantified in green propolis at 6.51%. Conclusions: The proposed method showed advantages in comparison with other methods, such as short analysis time and high selectivity for artepillin C.