Liquid Chromatography-electrospray Mass Spectrometry Research Articles

Liquid chromatography-electrospray ionization-tandem mass spectrometric assay for d-aspartate N-methyltransferase activity in ark shells.

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the separation and quantification of the enantiomers of N-methylaspartate and N-methylglutamate, after derivatization with Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide was established. The time required for the LC-ESI-MS/MS analysis was within 20min and the detection limit was approximately 10fmol per injection, demonstrating that this method can be used for the rapid determination of D-aspartate N-methyltransferase activity in the ark shell clam Scapharca broughtonii.Abbreviations: NMDA: N-methyl-D-aspartate; NMLA: N-methyl-L-aspartate; NMDG: N-methyl-D-glutamate; NMLG: N-methyl-L-glutamate; NMA: N-methylaspartate; NMG: N-methylglutamate; HPLC: high-performance liquid chromatography; SAM: S-adenosyl-L-methionine; OPA: o-phthalaldehyde; LC-ESI-MS/MS: liquid chromatography-electrospray ionization-tandem mass spectrometry; FDLA: Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide; FDAA: Nα-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide; ESI: electrospray ionization; LC-ESI-MS: liquid chromatography-electrospray ionization-mass spectrometry; MS/MS: tandem mass spectrometry.

Antioxidant Capacity of Thistle (Cirsium japonicum) in Various Drying Methods and their Protection Effect on Neuronal PC12 cells and Caenorhabditis elegans

The aim of this study was, firstly, to evaluate the phenol profile of thistle (Cirsium japonicum, CJ) by High performance liquid chromatography-electrospray ionization–mass spectrometry (HPLC–ESI–MS), dried by different methods (90 °C hot-air, 70 °C hot-air, shade-, and freeze-drying). Secondly, we aimed to evaluate the relationship between phenolic compounds content and antioxidant properties. CJ contained chlorogenic acid, linarin, and pectolinarin. Total phenolic contents of CJ significantly decreased under hot-air-drying condition, especially chlorogenic acid contents in CJ have been reduced by 85% and 60% for 90 °C and 70 °C hot-air-drying, respectively. We evaluated the protective effect on adrenal pheochromocytoma (PC12) cells and Caenorhabditis elegans using shade-dried CJ, which has the largest phenolic contents and the strongest antioxidant property. CJ-treated PC 12 cells dose-dependently exhibited the protective effects against reactive oxygen species (ROS), while cell viability increases, lactate dehydrogenase release decreases, and ROS formation decreases. Furthermore, CJ has also shown protection against ROS in C. elegans. Consequently, CJ contributed to lifespan extension under ROS stress without influencing the physiological growth.

Identification of secondary metabolites in Tripterygium wilfordii hairy roots and culture optimization for enhancing wilforgine and wilforine production

Hairy roots culture is an effective biological system to solve the shortage of natural resources of Tripterygium wilfordii for the production of the important secondary metabolites. However, there is no enough information about the secondary metabolites in Tripterygium wilfordii hairy root cultures. The present study identified the secondary metabolites in T. wilfordii hairy roots by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). Subsequently, the nitrogen source and phosphate concentration were optimized to enhance the production of the major active compounds wilforgine and wilforine. Nine compounds including triptolide, wilforgine, wilforine, and so on were identified according to their retention behavior in liquid chromatography and mass spectrometric fragmentation patterns. The Murashige and Skoog (MS) medium was modified for enhancing wilforgine and wilforine production in T. wilfordii hairy root cultures by altering the ratio of NH4+/NO3－and the concentration of phosphate. The results showed that 10:50 mM NH4+/NO3－ and 0.3125 mM phosphate were optimal for roots growth and the production of wilforgine and wilforine. After 35 days of cultivation in the modified MS medium containing 10:50 mM NH4+/NO3－ and 0.3125 mM phosphate, the maximum production of wilforgine and wilforine was up to 1509.87 μg·flask−1 and 578.56 μg·flask−1 in T. wilfordii hairy roots separately, a 42 % and 48 % increase over that in the basic MS medium. The present study laid a foundation for the large-scale production of secondary metabolites from T. wilfordii hairy roots.

Nickel(I) salen-catalyzed reduction of 1,1,2-trichloro-1,2,2-trifluoroethane (CFC-113): CO2-mediated carbon–fluorine bond cleavage

Cyclic voltammetry, controlled-potential (bulk) electrolysis, gas chromatography (GC), gas chromatography–mass spectrometry (GC–MS), and high-performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC–ESI–MS) have been used to investigate the direct and nickel(I) salen-catalyzed reduction of 1,1,2-trichloro-1,2,2-trifluoroethane (CFC-113) in both Ar- and CO2-saturated dimethylformamide (DMF) containing 0.10 M tetra-n-butylammonium tetrafluoroborate (TBABF4). Cyclic voltammograms show one irreversible cathodic peak for direct reduction of CFC-113 in CO2-saturated DMF to afford 1-chloro-1,2,2-trifluoroethene (CFC-1113), whereas cyclic voltammograms in Ar-saturated DMF exhibit two irreversible cathodic peaks. Bulk electrolyses of CFC-113 in CO2-saturated DMF at a potential 400 mV more negative than the resolved cathodic peak cause partial reduction of CFC-1113 to yield di-, tri-, and tetrafluoro species. Cyclic voltammograms for reduction of nickel(II) salen in the presence of CFC-113 in CO2-saturated DMF show two cathodic peaks attributable to formation of CFC-1113 and HFC-1123; cyclic voltammograms in Ar-saturated DMF exhibit similar behavior. Bulk electrolyses involving electrogenerated nickel(I) salen in CO2-saturated DMF yield mixtures of HFC-1123, isomers of difluoroethene, and traces of other species. No electrocarboxylation of CFC-113 or its derivatives was seen in the presence of CO2; however, excess CO2 increases the extent of dechlorination of CFC-113 during bulk electrolyses when compared with analogous electrolyses in an Ar-saturated medium. Structural modification of nickel(II) salen during catalytic reduction of CFC-113 in the presence of CO2 was determined with the aid of HPLC–ESI–MS.

Nutritional component analyses of kiwifruit in different development stages by metabolomic and transcriptomic approaches.

Metabolites in kiwifruit greatly influence nutritional values; however, the dynamic changes in nutrient composition and the gene expression level of yellow kiwifruit have not been studied so far. To investigate the types and accumulation patterns of metabolites, a metabolomics approach utilizing liquid chromatography-electrospray ionization mass spectrometry and transcriptomics were used to analyze the yellow flesh of kiwifruit cultivar 'jinshi 1' collected at different stages of days after full bloom. In total, 285 metabolites were identified over the kiwifruit developmental stages. The composition of the metabolites of kiwifruit at different stages of development was different. The organic acids contents and their derivatives were higher at the initial stage of development and then gradually decreased. The lipids and amino acids contents fluctuated at different stages of development but did not change significantly. Transcript profiles throughout yellow kiwifruit development were constructed and analyzed, with a focus on the biosynthesis and metabolism of compounds such as sugars, organic acids and ascorbic acid, which are indispensable for the development and formation of quality fruit. The transcript levels of genes involved in sucrose and starch metabolism were consistent with the change in soluble sugar and starch content throughout kiwifruit development. The metabolism of ascorbic acid was primarily through the l-galactose pathway. Our metabolome and transcriptome approach identified dynamic changes in five types of nutrient metabolite levels, and correlations among such levels, in developing fruit. The results provide information that can be used by metabolic engineers and molecular breeders to improve kiwifruit quality. © 2020 Society of Chemical Industry.

Lysine Reacts with Cholesterol Hydroperoxide to Form Secosterol Aldehydes and Lysine-Secosterol Aldehyde Adducts

Two cholesterol secosterol aldehydes, namely, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol A) and its aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxyaldehyde (secosterol B), are highly bioactive compounds which have been detected in human tissues and potentially contribute to the development of physiological dysfunctions such as atherosclerosis, Alzheimer’s disease, diabetes, and cancer. They were originally considered to be exclusive products of cholesterol ozonolysis and thus to be evidence for endogenous ozone formation. However, it was recently postulated that primary amines such as lysine may catalyse their formation from cholesterol-5α-hydroperoxide (Ch-5α-OOH), the main product of the oxidation of cholesterol with singlet oxygen. This involves cyclization of Ch-5α-OOH to an unstable dioxetane intermediate, which decomposes to form secosterol aldehydes with triplet carbonyl groups, whose return to the singlet state is at least partly coupled to the conversion of triplet molecular oxygen to singlet oxygen. Here, we subjected cholesterol to photosensitized oxidation, which predominantly produces Ch-5α-OOH and minor amounts of the 6α- and 6β-hydroperoxides, exposed the hydroperoxide mixture to lysine in the presence of the antioxidant 2,6-ditertiary-butyl-4-hydroxytoluene (BHT), and analysed the reaction mixture by liquid chromatography-electrospray ionization-mass spectrometry. Consistent with the postulated lysine-catalysed formation of secosterol aldehydes, we detected formation of the latter and several types of their lysine adducts, including carbinolamines, Schiff’s bases, and amide-type adducts. We propose that the amide type adducts, which are major biomarkers of lipid oxidation, are mainly formed by singlet oxygen-mediated oxidation of the carbinolamine adducts.

Using 4-(Carbazole-9-yl)-benzyl Chloroformate as a Derivatization Reagent for the Measurement of Amino Acids in Tea Samples by High-Performance Liquid Chromatography with Fluorescence Detection

An effective method for the determination of 20 free amino acids in tea samples using 4-(carbazole-9-yl)-benzyl chloroformate (CBBC-Cl) as a derivatization reagent by high-performance liquid chromatography combined with fluorescence detection (HPLC-FLD) has been established. CBBC-Cl quickly derivatized amino acids within 6 min in basic solution (pH 9.0) at room temperature (ca. 25 °C). The maximum excitation and emission wavelengths of the stable derivatives were 290 nm and 370 nm, respectively. The separation of 20 amino acids, including the theanine (THE) derivative, was optimized on a Hypersil BDS-C18 column. Identification of these derivatives was performed via liquid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) in positive-ion detection mode. Under optimal conditions, excellent linearity for all the amino acids was obtained, with determination coefficients of r2 > 0.9986. The detection limits (LODs) and quantitation limits (LOQs) ranged from 0.13 to 1.5 μg·kg−1 and 0.43 to 4.95 μg·kg−1, respectively. Satisfactory recoveries, ranging from 86.7 to 106.4%, were obtained for all the amino acids in the tea samples. The developed method has been successfully applied to the quantification of 20 amino acids in tea samples.

Changes in endogenous auxin level during flower bud abscission process in Pistachio (Pistacia vera L.)

The regulation of auxin (indole-3-acetic acid, IAA) level in plants is a complex mechanism and the concept is not completely understood. There are many lines of evidence suggesting that amino acid conjugates of IAA play a vital role in auxin homeostasis, but the involved plant enzymes are still under investigation. In the present study, the alteration in the levels of auxin and its conjugates in the different physiological stages and organs, as well as its role in flower bud abscission in Pistacia vera L., were investigated. The levels of the auxin and its conjugates, such as IAA, indole butyric acid (IBA), IAA-Ala, IAA-Leu, IAA-Asp, and IAA-Glu, were analyzed by ultraperformance liquid chromatography-electrospray ionization-mass spectrometry (UPLC/ESI-MS/MS) technique. Among the auxin conjugates, IAA was the main conjugate and dominant indole detected in pistachio extract observed in this work. It has also been determined that quite a few organs of 'Off-year' trees have higher auxin content than 'On-year' trees. It was observed that the auxin level is low in fruit-bearing trees in both leaves and shoots. It affects fruit yield and fruit bud abscission and plays some role in the alternate bearing. Decreased levels of auxin were observed in most of the organs of 'On-year' trees during kernel development resulting in bud abscission. This study shows that auxin conjugates play an important role in IAA metabolism, particularly in flower bud abscission and fruit kernel development.

Treatment with Synthetic Pseudoceramide Improves Atopic Skin, Switching the Ceramide Profile to a Healthy Skin Phenotype

Little is known about the pathophysiological linkages between altered ceramide profiles in the stratum corneum (SC) of patients with atopic dermatitis and their impaired skin barrier and water-holding functions. We studied those characteristics following topical treatment with a designed synthetic pseudoceramide (pCer) and analyzed that pathophysiological linkage by microanalyzing ceramides using normal phase liquid chromatography-electrospray ionization mass spectrometry. Four weeks of treatment with pCer significantly reduced skin symptoms, accompanied by significant decreases in transepidermal water loss and increases in water content. In the SC ceramide profiles, ceramides containing nonhydroxy fatty acids and 6-hydroxysphingosines (Cer[NH]) and ceramides containing nonhydroxy fatty acids and phytosphingosines (Cer[NP]) increased, whereas ceramides containing nonhydroxy fatty acids and sphingosines (Cer[NS]) and ceramides containing a-hydroxy fatty acids and sphingosines (Cer[AS]) decreased, with larger alkyl chain lengths in Cer[NS], distinctly representing a switch from an atopic dermatitis to a healthy skin phenotype. The level of pCer that penetrated into the SC was significantly correlated with the SC water content but not with transepidermal water loss. The level and the average carbon chain length of Cer[NS] were closely correlated with the pCer level in the SC. These findings indicate that the penetrated pCer contributes to shift the ceramide profile from an atopic dermatitis to a healthy skin phenotype. Taken together, the observed clinical efficacy of treatment with pCer provides a deep insight into the pathogenesis of atopic dermatitis as a ceramide-deficient disease.

Enzymatic Synthesis of 2-(β-Galactosyl)-ethyl Methacrylate by β-Galactosidase from Pyrococcus woesei and Application for Glycopolymer Synthesis and Lectin Studies.

Glycosidases have long been used for the synthesis of glycosides by transglycosylation reactions. Especially glycosidases from hyperthermophilic bacteria are useful for reactions under extreme reaction conditions, e.g., in the presence of organic solvents. We herein report the facile enzymatic synthesis and purification of 2-(β-galactosyl)-ethyl methacrylate (Gal-EMA) with the recombinant hyperthermostable glycosidase from Pyrococcus woesei in high yields. Optimized reaction conditions resulted in gram-scale synthesis of the galactosylated monomer with 88% transglycosylation yield. The product Gal-EMA was characterized by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS), nuclear magnetic resonance (NMR) spectroscopy, and infrared (IR) spectroscopy. Gal-EMA was utilized to synthesize sugar-functionalized acrylate polymers with defined amounts of incorporated galactose (0-100%). Analysis of the binding affinity of the lectin RCA120 from Ricinus communis to the glycopolymers using an enzyme-linked lectin assay (ELLA) revealed KD values between 0.24 and 6.2 nM, depending on the amount of incorporated Gal-EMA. The potential of Gal-EMA for the synthesis of acrylate-functionalized glycan oligomers was demonstrated by sequential elongation of the terminal galactose by two glycosyltransferases, resulting in the terminal glycan N-acetyllactosamine (LacNAc) epitope. In conclusion, the enzymatic synthesis of Gal-EMA opens new routes to a series of novel monomeric building blocks for the synthesis of glycan-functionalized polyacrylates.

Glycan biomarkers for Alzheimer disease correlate with T‐tau and P‐tau in cerebrospinal fluid in subjective cognitive impairment

Alzheimer disease (AD) is a devastating disease and a global health problem, and current treatments are only symptomatic. A wealth of clinical studies support that the disease starts to develop decades before the first symptoms appear, emphasizing the importance of studying early changes for improving early diagnosis and guiding toward novel treatment strategies. Protein glycosylation is altered in AD but it remains to be clarified why these alterations occur and how they affect the disease development. Here, we used a glycomics approach to search for alterations in protein glycosylation in cerebrospinal fluid (CSF) in AD compared with nondemented controls. Using both matrix‐assisted laser desorption ionization‐time of flight and liquid chromatography–electrospray mass spectrometry, we observed an increase in N‐glycans carrying bisecting N‐acetylglucosamine in AD. Based on those findings, we designed an enzyme‐linked multiwell plate assay to quantify N‐glycans binding to the lectin Phaseolus vulgaris Erythroagglutinin (PHA‐E), which is specific for N‐glycans containing bisecting N‐acetylglucosamine. Using this assay, we found a similar increase in CSF in AD compared with controls. Further analysis of CSF from 242 patients with subjective cognitive impairment (SCI), mild cognitive impairment (MCI), or AD dementia revealed significantly increased binding to PHA‐E in MCI and AD compared to SCI. Interestingly, PHA‐E binding correlated with CSF levels of phosphorylated tau and total tau and this correlation was most prominent in the SCI group (R = 0.53–0.54). This study supports a link between N‐glycosylation, neurodegeneration, and tau pathology in AD and suggests that glycan biomarkers have potential to identify SCI cases at risk of developing AD.

A subcellular proteome atlas of the yeast Komagataella phaffii.

ABSTRACTThe compartmentalization of metabolic and regulatory pathways is a common pattern of living organisms. Eukaryotic cells are subdivided into several organelles enclosed by lipid membranes. Organelle proteomes define their functions. Yeasts, as simple eukaryotic single cell organisms, are valuable models for higher eukaryotes and frequently used for biotechnological applications. While the subcellular distribution of proteins is well studied in Saccharomyces cerevisiae, this is not the case for other yeasts like Komagataella phaffii (syn. Pichia pastoris). Different to most well-studied yeasts, K. phaffii can grow on methanol, which provides specific features for production of heterologous proteins and as a model for peroxisome biology. We isolated microsomes, very early Golgi, early Golgi, plasma membrane, vacuole, cytosol, peroxisomes and mitochondria of K. phaffii from glucose- and methanol-grown cultures, quantified their proteomes by liquid chromatography-electrospray ionization-mass spectrometry of either unlabeled or tandem mass tag-labeled samples. Classification of the proteins by their relative enrichment, allowed the separation of enriched proteins from potential contaminants in all cellular compartments except the peroxisomes. We discuss differences to S. cerevisiae, outline organelle specific findings and the major metabolic pathways and provide an interactive map of the subcellular localization of proteins in K. phaffii.

Is 9β-dehydrohalogenation of betamethasone and dexamethasone hindering the detection of banned co-eluting meprednisone? A reverse-phase chiral liquid chromatography high-resolution mass spectrometry approach

Mass spectral analysis of dexamethasone and betamethasone reveal intense signals at m/z 373.19994 (using a Thermo Q Exactive high-resolution mass spectrometer coupled with Dionex UltiMate 3000 UHPLC + operated in the positive ion mode), matching the signal of meprednisone, the 11-oxo version of methylprednisolone, along with its parent signal; possibly due to dehydrohalogenation of these drugs at MS. The parent mass of meprednisone is exactly same as that of dehydrohalogenated mass of dexamethasone and betamethasone; and are co-eluting, displaying same mass spectra. Specifically when they are administered together, identifying meprednisone (a drug for which there is zero tolerance in some regions of the world), is a great challenge with currently available techniques because it could be easily mistaken for dexamethasone or betamethasone, drugs allowed at certain threshold limits for therapeutic considerations. False negative results could be obtained in conventional reverse-phase chromatography and are liable to be abused; hence, establishing “zero tolerance” limits for these compounds often proves ineffective. In this paper, present an effective and reliable analytical method for simultaneously separating and identifying dexamethasone, betamethasone and meprednisone in equine urine and plasma using chiral liquid chromatography-electrospray ionization-mass spectrometry. From the various columns screened, the Lux i-Cellulose-5 chiral column produced high-quality results with extremely good separation. During this study, it is quite evident that dehydrohalogenation occurs only in the mass ionization source; the compounds are very stable in-vivo/in-vitro and do not break down either on-column or during sample preparation.

Antidementia effects, metabolic profiles and pharmacokinetics of GJ-4, a crocin-rich botanical candidate from Gardeniae fructus.

Crocins, a series of hydrophilic carotenoids that are either mono- or di-glycosyl polyene esters of crocetin extracted from dried saffron stigma or fruits of gardenia, are attracting much attention due to their wide range of pharmacological effects. In our previous study, GJ-4, a mixture of crocin analogues, was obtained and derived from gardenia fruits. Mainly 18 crocin analogues were identified from GJ-4 and found to exhibit neuroprotective effects in in vitro and in vivo models. In this present study, we continue to investigate the therapeutic effects of GJ-4 on learning and memory impairments in a 2VO-induced VaD model, and the potential mechanism. In addition, the metabolic profiles and pharmacokinetic properties of GJ-4 were determined using liquid chromatography-electrospray ionization-mass spectrometry after single and multiple oral doses. All these findings presented here will serve as a solid basis to develop GJ-4 as a new therapeutic agent for dementia.

A Novel LC-MS/MS Method Development and Validation for Ultra-trace Level Determination of Three Genotoxic Efavirenz Impurities

A novel, simple, sensitive, specific and rapid reverse-phase liquid chromatography-electrospray ionization-mass spectrometry method was established and validated for the ultra-trace analysis of efavirenz-related compound C, 8-hydroxy efavirenz and its impurity 1 which are potential genotoxic impurities associated with efavirenz. These were separated on high performance liquid chromatography and detected with tandem mass spectrometry in the positive ionization mode with MRM transitions, m/z 314.15>244.10, 212.10>57.10, 305.70>69.05 and 364.15>291.25. A gradient method was developed to separate the impurities using 0.01 M ammonium acetate buffer and methanol at 1 ml/min flow-rate. Samples were analyzed using a Hypersil C-18 column (5 µm, 250×4.6 mm). The calibration curves have shown good linearity in the concentration range of 2.5-78 ppb and a correlation coefficient of 0.99. Mean intra and inter day precision was 2.6-3.2 ppm and 2.5-2.6 ppm, respectively. For the 3 genotoxic impurities the limit of detection and limit of quantitation were 0.04 ppm and 0.125 ppm, respectively. The liquid chromatography-mass spectrometry/mass spectrometry method developed was specific, sensitive, precise and accurate. The developed method could be used for quantification and monitoring of genotoxic impurities of efavirenz.

Early detection of metabolic changes in drug-induced steatosis using metabolomics approaches.

Steatosis is the accumulation of triglycerides in hepatic cells wherein fats exceed 5% of the entire liver weight. Although steatotic liver damage is reversible due to the liver's regenerative capability, protracted damage often and typically leads to irreversible conditions such as cirrhosis and hepatocellular carcinoma (HCC). Therefore, early steatotic detection is critical for preventing progression to advanced liver diseases. This also becomes particularly important given the higher prevalence of drug usage, as drugs are a frequent cause of liver damage. Currently, the recommendation to diagnose steatosis is using liver enzymes and performing a liver biopsy. Liver biopsy remains the gold standard method of detection, but the procedure is invasive and an unreliable diagnostic tool. Non-invasive, specific and sensitive diagnostic solutions such as biomarkers are therefore needed for the early detection of steatosis. Our aim is to identify changes in urinary metabolites in tetracycline-induced hepatic steatotic rats at different stages of the diseases using metabolomic-based techniques. Sprague Dawley male rats are treated by intraperitoneal injection (I.P.) with either 62.5 mg kg−1 or 125 mg kg−1 tetracycline, an antibiotic previously known to induce steatosis. We analyse the metabolic profile of the urinary tetracycline induced hepatic steatotic rats using 1H nuclear magnetic resonance (NMR), 2D 1H–1H TOCSY (total correlation spectroscopy) and electrospray liquid chromatography-mass spectrometry (ESI-LC-MS/MS) based metabolomics. The combined analysis of haematoxylin & eosin (H&E), oil red O (ORO) and direct measurement of triglyceride content in the liver tissues of the control samples against 125 mg kg−1 and 62.5 mg kg−1 treated samples, reveals that 125 mg kg−1 tetracycline exposure potentially induces steatosis. The combination of 1H NMR, 2D 1H–1H TOCSY and ESI-LC-MS/MS alongside multivariate statistical analysis, detected a total of 6 urinary metabolites changes, across 6 metabolic pathways. Furthermore, lysine concentration correlates with liver damage as tetracycline dose concentration increases, whilst both H&E and ORO fail to detect hepatocellular damage at the lowest dose concentration. We conclude that the combination of 1H NMR and ESI-LC-MS/MS suggests that these are suitable platforms for studying the pathogenesis of steatosis development, prior to morphological alterations observed in staining techniques and offer a more detailed description of the severity of the steatotic disease.

Acute Toxicity of the Hydroethanolic Extract of the Flowers of Acmella oleracea L. in Zebrafish (Danio rerio): Behavioral and Histopathological Studies.

Hydroethanolic preparations of the botanical species Acmella oleracea L. are used in the north of Brazil for the treatment of various diseases. However, few studies have been conducted to evaluate the toxicity of this species. The objective of this study was to evaluate the acute toxicity of the hydroethanolic extract of A. oleracea L. (EHFAo) flowers in zebrafish by immersion and oral administration. The extract was analyzed by ultra-performance liquid chromatography–mass spectrometry (UPLC–MS). EHFAo was administered orally (44.457, 88.915, 199.94, 281.83, and 448.81 mg/kg) and by immersion (250, 300, 350, 400, and 450 µg/L). Behavioral and histopathological analysis of gills, liver, intestine, and kidney were performed. The presence of (2E,6Z,8E)-N-isobutyl-2,6,8-decatrienamide (spilanthol) in EHFAo was identified by ultra-high-re.solution liquid chromatography–electrospray ionization mass spectrometry (UHPLC–ESI-MS). Treatment with EHFAo caused significant behavioral changes and death. The calculated median lethal dose (LD50) was 148.42 mg/kg, and the calculated median lethal concentration (LC50) was 320 μg/L. In the histopathological study, it was observed that upon oral treatment, the tissue alterations that compromised the normal functioning of the organism occurred with EHFAo doses of 88.915, 199.53, and 281.83 mg/kg, the intestine being the most affected. When the treatment was performed by immersion, the most toxic EHFAo concentrations according to the histopathological evaluation were 300, 350, and 400 μg/L, with the most affected organ being the gills. Finally, EHFAo in this study was shown to be more toxic to the liver, intestine, and kidneys when administered orally and to gills, liver, and kidneys when administered by immersion in water. Therefore, considering the results obtained and the chemical characteristics of the main phytochemical marker of EHFAo, spilanthol, it can be suggested that, depending on the dose, this compound can lead to histopathological damages in the organs highlighted in this study.

A comparative metabolomics analysis of the components of heartwood and sapwood in Taxus chinensis (Pilger) Rehd.

Taxus chinensis is a well-known gymnosperm with great ornamental and medicinal value. Its purple red brown heartwood (HW) has many attributes such as straight texture, high density, mechanical strength, rich elasticity and corrosion resistance that is highly prized commercially. T. chinensis sapwood (SW), in comparison, lacks these important traits. At present, little is known about the differences of metabolites between the SW and HW in T. chinensis. Widely targeted metabolic profiling was performed to analyze the metabolic profiles of HW and SW in T. chinensis using Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-EI-MS). A total of 607 metabolites were detected in HW and SW. Among them, 146 metabolites were significantly higher, and 167 metabolites significantly lower, in HW as compared to SW. These differential metabolites were mainly involved in metabolic pathways and biosynthesis of secondary metabolites, such as flavonoids, flavone and flavonol, phenylpropanoids and antibiotics. Moreover, 71 flavonoids and isoflavones were found to be significantly different between HW and SW. Our results show the difference of components between the HW and SW, which has potential significance to further elucidate the mechanism of HW color formation. The results will provide insight into the metabolites associated with wood color formation and useful information for understanding the metabolites associated with wood quality.

Dynamic distribution of azoxystrobin residues in strawberry (Fragaria x ananassa Duchesne) using liquid chromatography tandem mass spectrometry: Putative evaluation of dietary intake

ABSTRACT A simple, rapid and environmentally compatible method involving quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction technique for simultaneous detection of azoxystrobin residues in strawberry was optimised and validated. Liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) was also used for quantitative and confirmatory analysis of azoxystrobin residues in strawberry. Recoveries ranged between 96% and 111% and repeatability (expressed as percentage relative standard deviation, % RSD) typically <11% has been well achieved. The dissipation pattern of azoxystrobin in different treatments level, based on the most critical field use patterns was reported utilising the method developed in this study. The results revealed that the degradation of azoxystrobin in strawberry followed first-order kinetics (R2 > 0.974) with estimated half-life value of 8.45 days. Considering the terminal residues and the maximum residue limit (MRL, 10 mg kg−1) in strawberry, and based on this study, it is safe to harvest strawberry 3 days after applying azoxystrobin even at rated higher the recommended one with exaggerated, suggesting that the use of this compound is safe for humans. The results could provide a guidance to fully evaluate the risks of azoxystrobin to consumers.

Momilactones A, B, and Tricin in Rice Grain and By-Products are Potential Skin Aging Inhibitors.

We previously reported the inhibitory potentials of momilactones A (MA) and B (MB) against key enzymes related to type 2 diabetes and obesity. In this study, antioxidant and anti-skin-aging activities of MA and MB were investigated and compared with tricin, a well-known antioxidant and antiaging flavonoid in rice. MA, MB, and tricin were purified from rice husk by column chromatography and their biological activities were subsequently assayed by in vitro trials. The contents of MA, MB, and tricin of different commercial rice cultivars in Japan were quantified and confirmed by ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) and high-performance liquid chromatography (HPLC) analyses. The antioxidant assays revealed a synergistic activity of the mixture MA and MB (MAB, 1:1, v/v). In addition, in 2,2’-azino-bis (ABTS) assay, IC50 values of MAB (0.3 mg/mL) and tricin (0.3 mg/mL) was 4-fold and 9-fold greater than that of individual MB (1.3 mg/mL) or MA (2.8 mg/mL), respectively. The in vitro enzymatic assays on pancreatic elastase and tyrosinase indicated that MA and MB were potential to relief skin wrinkles and freckles. In detail, MA exerted higher inhibition on both enzymatic activities (30.9 and 37.6% for elastase and tyrosinase inhibition, respectively) than MB (18.5 and 12.6%) and MAB (32.0 and 19.7%) at a concentration of 2.0 mg/mL. Notably, MA and the mixture MAB exhibited stronger inhibitions on elastase and tyrosinase in comparison with tricin and vanillin. MA, MB, and tricin in rice are potential to develop cosmetics as well as supplements for skin aging treatments.