Time Of Flight Tandem Mass Spectrometry Research Articles

Antimicrobial activities of polyphenol-based metabolites present in lettuce and recent methods for their estimation

The tissue structure of fresh-cut lettuce is easy to be damaged during processing and transportation, which leads to the reduction of its edible quality and safety. Ultra-high-performance liquid chromatography (UHPLC) combined with time-of-flight tandem mass spectrometry (TMS) metabolomics was used to identify the differential metabolites related to the antibacterial activity in lettuce and explore their bacteriostatic mechanism. The experimental results revealed that the growth of Escherichia coli was quite different when different varieties of lettuce were used as the nutrient substrate. Between the varieties Beizisheng No. 3 (BZ3) and Shooter 101 (SS), 204 differential metabolites showed significant changes (P < 0.05), 86 metabolites showed changes between the varieties BZ3 and Beisansheng No. 1 (BS1). Among the metabolites, isoquercitrin was the upregulated differential metabolite in BZ3 compared to SS and BS1. The content of isoquercitrin in lettuce juice positively correlated with the antibacterial activity of polyphenol extract, but negatively correlated with the growth of E. coli. The bacteriostatic property of polyphenol extract destroys the morphological structure of E. coli. The higher the isoquercitrin content, the stronger the resistance of fresh-cut lettuce to E. coli.

Comparative Study on Growth and Metabolomic Profiles of Six Lactobacilli Strains by Sodium Selenite

Selenium (Se) has garnered increasing attention in the field of nutrition, as it is essential for both humans and animals. Certain microorganisms can enrich inorganic selenium and convert it into organic selenium. The growth and metabolomic profiles of six lactobacilli strains exposed to 50 μg/mL of sodium selenite were performed using gas chromatography tandem time-off light mass spectrometry (GC-TOF-MS) analysis. The addition of selenium significantly increased both the population and weight of the Lacticaseibacillus rhamnosus PS5, Lbs. rhamnosus RT-B, Limosilactobacillus reuteri 3630, and Lmb. reuteri 1663 strains, while those of the other two strains decreased. A total of 271 metabolites were determined, with their concentrations ranked from highest to lowest as follows: organic acids and derivatives, oxygen compounds, lipids and lipid-like molecules, and benzenoids. In certain groups, the concentrations of serine, aspartic acid, trehalose, palmitic acid, methylthreonine, and melibiose increased significantly, whereas glucuronic acid, ribose, ornithine, and methionine were downregulated. The metabolic pathways were significantly associated with ABC transporters, glycine, serine, threonine metabolism, and aminobenzoate degradation and other pathways. Based on these findings, we concluded that the transport, absorption, assimilation, and stress response to selenium by lactobacilli in metabolomic changed. Furthermore, the metabolomic alterations among different types of lactobacilli varied primarily due to their distinct properties.

Thevetia thevetioides Cardenolide and Related Cardiac Glycoside Profile in Mature and Immature Seeds by High-Resolution Thin-Layer Chromatography (HPTLC) and Quadrupole Time of Flight-Tandem Mass Spectrometry (Q-TOF MS/MS) Reveals Insights of the Cardenolide Biosynthetic Pathway.

Thevetia thevetioides is a species within the Apocynaceae family known for containing cardenolide-glycosides, commonly referred to as cardiac glycosides, which are characteristic of this genus. The seeds of the Thevetia species are frequently used as a model source for studying cardiac steroids, as these glycosides can be more readily extracted from the oil-rich seeds than from the plant's green tissues. In this work, the cardenolide profile of ripe and immature seeds was determined and compared to establish the main differences. Ripe seeds contain six related cardenolides and triosides, with thevetin B being the predominant component. In contrast, immature seeds exhibit a total of thirteen cardiac glycosides, including monoglycosides such as neriifolin and peruvosides A, B, and C, as well as diglycosides like thevebiosides A, B, and C. Some of these compounds have previously been identified as degradation products of more complex cardiac glycosides; however, their presence in immature seeds, as described in this study, suggests that they may serve as biosynthetic precursors to the triosides observed in mature seeds. The glycoside patterns observed via HPTLC are associated with specific chemical structures characteristic of this genus, typically featuring thevetose or acetyl-thevetose at the first position, followed by glucose or gentibiose in di- or trisaccharides, independent of the trioside aglycones identified: digitoxigenin, cannogenin, or yccotligenin. Ripe seeds predominantly contain triosides, including thevetin B, C, and A, the latter of which has not been previously reported.

Discovery, isolation and structural identification of alkaloid glycosides in six traditional Chinese medicine such as Coptis chinensis

Alkaloids are important active ingredients occurring in many traditional Chinese medicines, and alkaloid glycosides are one of their existence forms. The introduction of saccharide units improves the water solubility of alkaloid glycosides thus presenting better biological activity.Because of the low content in plants, alkaloid glycosides have been not comprehensively studied. In this study, ultrahigh performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry(UPLC-QTOF-MS/MS) was employed to identify and analyze the alkaloid glycosides in Coptis chinensis, Phellodendron chinense, Menispermum dauricum, Sinomenium acutum, Tinospora sagittata and Stephania tetrandra. The results showed that except Tinospora sagittata, the other five herbal medicines contained alkaloid glycosides. Furthermore, the alkaloid glycosides in each herbal medicine were identified based on UV absorption spectra, quasimolecular ion peaks in MS, fragment ions information in the MS/MS, and previous literature reports. A total of 42 alkaloid glycosides were identified. More alkaloid glycosides were identified in C. chinensis and Menispermum dauricum, and eleven in C. chinensis were potential new compounds. Furthermore, the alkaloid glycosides in the water extract of C. chinensis were coarsely se-parated by macroporous adsorption resin, purified by column chromatography with D151 cation exchange resin, ODS and MCI, combined with semi-preparative high performance liquid chromatography. Two new alkaloid glycosides were obtained, and their structures were identified by mass spectrometry and NMR data as(S)-7-hydroxy-1-(p-hydroxybenzyl)-2,2-N,N-dimethyl-1,2,3,4-tetrahydroisoquinoline-6-O-β-D-glucopyranoside and(S)-N-methyltetrahydropalmatubine-9-O-β-D-glucopyranoside, respectively. This study is of great significance for enriching the information about the chemical composition and the in-depth development of C. chinensis. Meanwhile, it can provide a reference for rapid identification and isolation of alkaloid glycosides from other Chinese herbal medicines.

6-Oxofurostane and (iso)Spirostane Types of Saponins in Smilax sieboldii: UHPLC-QToF-MS/MS and GNPS-Molecular Networking Approach for the Rapid Dereplication and Biodistribution of Specialized Metabolites.

Identifying novel phytochemical secondary metabolites following classical pharmacognostic investigations is tedious and often involves repetitive chromatographic efforts. During the past decade, Ultra-High Performance Liquid Chromatography-Quadrupole Time of Flight-Tandem Mass Spectrometry (UHPLC-QToF-MS/MS), in combination with molecular networking, has been successfully demonstrated for the rapid dereplication of novel natural products in complex mixtures. As a logical application of such innovative tools in botanical research, more than 40 unique 3-oxy-, 3, 6-dioxy-, and 3, 6, 27-trioxy-steroidal saponins were identified in aerial parts and rhizomes of botanically verified Smilax sieboldii. Tandem mass diagnostic fragmentation patterns of aglycones, diosgenin, sarsasapogenin/tigogenin, or laxogenin were critical to establishing the unique nodes belonging to six groups of nineteen unknown steroidal saponins identified in S. sieboldii. Mass fragmentation analysis resulted in the identification of 6-hydroxy sapogenins, believed to be key precursors in the biogenesis of characteristic smilaxins and sieboldins, along with other saponins identified within S. sieboldii. These analytes' relative biodistribution and characteristic molecular networking profiles were established by analyzing the leaf, stem, and root/rhizome of S. sieboldii. Deducing such profiles is anticipated to aid the overall product integrity of botanical dietary supplements while avoiding tedious pharmacognostic investigations and helping identify exogenous components within the finished products.

Integrated 16S rRNA Gene Sequencing and Metabolomics Analysis to Investigate the Important Role of Osthole on Gut Microbiota and Serum Metabolites in Neuropathic Pain Mice.

Accumulating evidence suggests that neuropathic pain (NP) is closely connected to the metabolic disorder of gut microbiota, and natural products could relieve NP by regulating gut microbiota. The purpose of this study is to investigate the important regulatory effects of osthole on gut microbiota and serum metabolites in mice with chronic constriction injury (CCI). Mice’s intestinal contents and serum metabolites were collected from the sham group, CCI group, and osthole treatment CCI group. The 16S rRNA gene sequencing was analyzed, based on Illumina NovaSeq platform, and ANOVA analysis were used to analyze the composition variety and screen differential expression of intestinal bacteria in the three groups. Ultra-high-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry (UHPLC-Q-TOF-MS) was used for analyzing the data obtained from serum specimens, and KEGG enrichment analysis was used to identify pathways of differential metabolites in the treatment of neuralgia mice. Furthermore, the Pearson method and Cytoscape soft were used to analyze the correlation network of differential metabolites, gut microbiota, and disease genes. The analysis results of 16S rRNA gene sequencing displayed that Bacteroidetes, Firmicutes, and Verrucomicrobia were highly correlated with NP after osthole treatment at the phylum level. Akkermansia, Lachnospiraceae_unclassified, Lachnospiraceae_NK4A136_group, Bacteroides, Lactobacillus, and Clostridiales_unclassified exhibited higher relative abundance and were considered important microbial members at genus level in neuralgia mice. Serum metabolomics results showed that 131 metabolites were considered to be significantly different in the CCI group compared to the sham group, and 44 metabolites were significantly expressed between the osthole treatment group and the CCI group. At the same time, we found that 29 differential metabolites in the two comparison groups were overlapping. Integrated analysis results showed that many intestinal microorganisms and metabolites have a strong positive correlation. The correlation network diagram displays that 10 genes were involved in the process of osthole alleviating NP through a metabolic pathway and gut microbiota, including IGF2, GDAP1, MYLK, IL18, CD55, MIR331, FHIT, F3, ERBB4, and ITGB3. Our findings have preliminarily confirmed that NP is closely related to metabolism and intestinal microbial imbalance, and osthole can improve the metabolic disorder of NP by acting on gut microbiota.

Structure elucidation of new kinds of bacitracin sulfonic acid based on high performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry

Bacitracin is a mixture of antimicrobial peptides. Although the structures of bacitracins have been fully studied, the structure elucidation of the oxidation products of bacitracin has rarely been reported. In the present work, the bacitracin was treated by hydrogen peroxide solution to form bacitracin sulfonic acids (SO3H) directly, and the structures of bacitracin-SO3Hs were studied by high performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry (HPLC-QTOF-MS/MS). It was demonstrated that the components of bacitracin, such as bacitracin A, B, and C could be site-specifically transformed to the corresponding oxidized product containing the SO3H group. The structure characterization of the oxidized products was elucidated based on the accurate molecular mass and the product ions obtained by MS/MS. Some new kinds of bacitracin J-SO3H, vinyl-bacitracin A-SO3H, bacitracin Y–SO3H and deamino-bacitracin Y–SO3H were identified. Furthermore, we also investigated the novel fragmentation behavior of the oxidized products. This is the first report to propose a neutral loss mechanism of SO3 that explains the characteristic ions of the vinyl-bacitracin A-SO3H and bacitracin Y–SO3H. Based on the structure analysis of the oxidation products of bacitracin, three transformation routes of bacitracin-SO3H generated from bacitracin were hypothesized and systematically summarized for the first time.

Untargeted profiling of field cultivated bush tea (Athrixia phylicoides DC.) based on metabolite analysis

Bush tea (Athrixia phylicoides DC.) is an aromatic South African indigenous plant used for many decades as a health beverage and medicine. Several studies have extensively investigated wild bush tea's secondary metabolites, but the entire profiling of cultivated bush tea's metabolites is limited in the literature. Thus, the objective of this study was to profile cultivated bush tea metabolites using liquid chromatography-quadrupole time of flight-tandem mass spectrometry (LC-QTOF-MS). The 31 metabolites profiled included; benjaminamide, chlorogenate, chrysosplenetin, coumarin, 6Z-docosenamide, naringenin 7-O-β-d-glucoside, 5-p-coumaroylquinic acid, integrastatin A, luteolin 7-O-(6-O-malonyl-β-d-glucoside), 1,3-dicaffeoylquinic acid, magnoshinin, okanin, (2S)-5-hydroxy-7-methoxy-6,8-dimethylflavanone, (9Z,12Z,15Z)-octadecatrienoic acid, 2″-deamino-2″-hydroxy-6″-dehydroparomamine, O-butanoylcarnitine, myricitrin, gorlic acid, tetracenomycin X, sakuranin, d-tryptophan, linoleamide, laricitrin 7-monoglucoside, l-β-phenylalanine, l-proline, pheophytin A, pheophorbide A, PI(18:0/20:4(8Z,11Z,14Z,17Z), stearidonic acid, and gibberellin A14 aldehyde. These annotated metabolites included phenolics, flavonoids, and quinic acids, indicating that bush tea is rich in metabolites, which have a potential wide range of health benefits.

Characterizing metabolites and potential metabolic pathways changes to understanding the mechanism of medicinal plant Phellodendri Amurensis cortex against doxorubicin-induced nephritis rats using UPLC-Q/TOF-MS metabolomics

Phellodendri Amurensis cortex (PAC), a famous traditional Chinese herb with good anti-inflammatory efficacy, is used to treat various liver and kidney sickness in clinical practice. However, the potential mechanisms protecting against nephritis of PAC have not been comprehensively elucidated. The aim of this research was to explore the mechanism of PAC against doxorubicin- induced nephritis in rats by characterizing metabolites and potential metabolic pathways changes. The rat models of nephritis were established using 6.5 mg/kg doxorubicin injection from caudal vein for five weeks. The rats in the treatment group were respectively received PAC extract at the dose of 216, 432, and 864 mg/kg once a day during the experiment. Then, urine metabolomics strategy based on ultra-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry (UPLC-Q/TOF-MS) has been employed to discover the possible significant metabolites and metabolic pathway of nephritis rats. At the end of the experiment, serum, urine and kidney tissue were collected for biochemical and pathological examination. The results showed that PAC treatment notably decreased urinary protein, serum Cr content and renal tissue lesions, and increased serum TP and ALB content. A total of potential twenty- eight metabolites such as 5'-methylthioadenosine, cGMP, dehydroepiandrosterone sulfate, salbuta, 2-phenylaminoadenosine contributing to nephritis rat model were selected and identified in the urine samples. Compared with the model group, the high-dose PAC group can recall 18 metabolites level, the medium-dose group can recall 13 metabolites level, and the low-dose PAC group can recall 8 metabolites level, which were involved in nine primary metabolic pathways such as steroid hormone biosynthesis, alanine,aspartate and glutamate metabolism, cysteine and methionine metabolism as well as glyoxylate and dicarboxylate metabolism. The protein expressions of key enzymes involving methylthioadenosine phosphorylase (Mtap), cytidine deaminase (Cda), thymidine kinase (Tk), argininosuccinate synthase (Ass) in metabolic pathways were further verified by Western blot. The results showed that Phellodendron chinense up-regulated the protein expressions of Cda and Tk and down-regulated the protein expressions of Mtap and Ass. In conclusion, PAC possesses renoprotective effect against doxorubicin-induced nephritis, which may be mediated via regulating differential metabolites, reducing oxidative stress response, improving renal function, enhancing the ability of the immune system, regulating the role of key enzymes.

A novel determination method for diuron in seaweed samples: Combination of quadruple isotope dilution strategy with liquid chromatography - quadrupole time of flight - tandem mass spectrometry for superior accuracy and precision

This paper proposed an accurate and sensitive analytical method based on the combination of Liquid Chromatography-Quadrupole Time of Flight-Tandem Mass Spectrometry (LC-QTOF-MS/MS) system with Quadruple Isotope Dilution-Mass Spectrometry (ID4MS) strategy for the determination of diuron at trace levels. In order to achieve accurate and reliable determination of the analyte, ID4MS strategy that uses stable isotopically labelled analogues was employed. Diuron-d6 was synthesized in the laboratory using a novel strategy and employed in ID4MS for the preparation of three calibration blends and seaweed sample blend. The analytical performance of the LC-QTOF-MS/MS method was evaluated and the respective detection and quantification limits obtained were 14.6 µg kg−1 and 46.5 µg kg−1. Good linearity was obtained with a correlation coefficient of 0.9995 over the dynamic range from 50 to 1000 µg kg−1. The validity of the method was successfully tested by carrying out spiking experiments in seaweed samples. The percent recovery value showed superior enhancement for ID4MS strategy (99.97 ± 0.41%) confirming the applicability of the method to complex matrices with great accuracy and precision.

Health risk associated with the oral consumption of “Chiniy-tref”, a traditional medicinal preparation used in Martinique (French West Indies): Qualitative and quantitative analyses of aristolochic acids contained therein

“Chiniy-tref” (CT) is a traditional preparation used in folk medicine in Martinique Island (French West Indies) that is nowadays mainly taken orally to prevent or act against any “manifestation of evil”. CT is easily prepared at home by macerating larvae of the endemic swallowtail Battus polydamas (ssp.) cebriones (Dalman, 1823), sometimes accompanied by a leaf of its host-plant Aristolochia trilobata L., in commercial rum. We have previously reported the detection of nephrotoxic and carcinogenic aristolochic acids (AAs) I and II in CT, leading the Regional Health Agency (ARS) of Martinique to issue an alert regarding the potential risks associated with its consumption in 2015. In order to complete the toxicity risk assessment for oral consumption of CT, a full qualitative analysis of AAs and their analogues (AAAs) was performed, as well as a quantitative determination of the major AAs, namely AAs I and II. The phytochemical profiling of AAAs present in CT, that also corresponds to that of B. polydamas cebriones larvae feeding on A. trilobata, has been established for the first time by ultra-high performance liquid chromatography/electrospray ionization quadrupole time of-flight tandem mass spectrometry. AAs I and II were quantified in a small panel of tinctures by using a validated UHPLC/UV method, allowing us to estimate the probable daily intakes of these toxins by CT consumers. The results proved the existence of a real risk of renal toxicity and carcinogenicity associated with the chronic oral consumption of CT in Martinique, and more generally of similar “snake bottles” throughout the Caribbean.

Determination of glycine in body fluids at trace levels using the combination of quadrupole isotope dilution strategy and Liquid Chromatography-Quadrupole Time of Flight-Tandem Mass Spectrometry

In this study, Quadrupole Isotope Dilution Mass Spectrometry (ID4MS) strategy was coupled with Liquid Chromatography-Quadrupole Time of Flight-Tandem Mass Spectrometry (LC-QTOF-MS/MS) for the accurate and precise determination of glycine in body fluids at trace levels. The instrumental variables such as column temperature, injection volume, sample/gas flow rates, fragmentor/capillary voltages, were optimized. Under the all optimized conditions, the calibration plot of glycine demonstrated a linearity ranging 5.0–100 mg kg−1 and, the detection and quantification limits were obtained as 1.4 and 4.7 mg kg−1, respectively. Superior enhancement was achieved for the accuracy and precision of proposed method when combined with ID4MS strategy. Percent recoveries of the proposed method for human plasma and urine spiked at 80 mg kg−1 were calculated as 99.72 ± 0.54% and 100.91 ± 1.32%, respectively. These results validated the applicability of the method to complex matrices as well as confirming the high accuracy and precision.

Comprehensive Analysis of Secondary Metabolites in Usnea longissima (Lichenized Ascomycetes, Parmeliaceae) Using UPLC-ESI-QTOF-MS/MS and Pro-Apoptotic Activity of Barbatic Acid

Considering the importance of ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-tandem mass spectrometry (UPLC-ESI-QTOF-MS/MS) hyphenated techniques for analysis of secondary metabolites from crude extracts, the present study was aimed at identification of secondary metabolites in acetone extract of the lichen Usnea longissima. From our study, 19 compounds were tentatively identified through comparison of exact molecular masses from their MS/MS spectra, mass fragmentation studies and comparison with literature data. In addition, potent cytotoxic activity of U. longissima extract prompted us to isolate four compounds, 18R-hydroxy-dihydroalloprotolichesterinic acid (19), neuropogolic acid (20), barbatic acid (21), and usnic acid (22) from this extract which were adequately identified through mass spectrometry and NMR spectroscopy. All four compounds displayed cytotoxic activity. Barbatic acid (21) manifested doxorubicin equivalent activity against A549 lung cancer cell line with IC50 of 1.78 µM and strong G0/G1 accumulation of cells. Poly ADP-ribose polymerase (PARP) cleavage confirmed that it induced cytotoxic activity via apoptosis. Finally, our work has discerned the depside, barbatic acid (21) from crude extract as a candidate anti-cancer molecule, which induces cell death by stepping up apoptosis.

Rapid discrimination and quantification of isomeric flavonoid-O-diglycosides in Citrus paradisi cv. changshanhuyou by online extraction–quadrupole time-of flight tandem mass spectrometry

Rapid differentiation, characterization and quantification of isomers from complex mixtures by direct mass spectrometry (MS) remained an analytical challenge due to their similar or identical MS/MS spectra and matrix interferences. Here, we reported a novel online extraction–quadrupole time-of-flight tandem mass spectrometry (OLE–QTOF-MS/MS) system to rapid, efficient and sensitive analysis of interglycosidic linkage isomers (hesperidin and neohesperidin) in Citrus paradisi cv. Changshanhuyou (Changshanhuyou). OLE system packed with solid Changshanhuyou (0.02 mg) could be firstly used to online remove interferences with large polarities, and then online extract and enrich hesperidin and neohesperidin, which shows great potential to diminish the analysis time of sample pretreatment, as well as to reduce matrix effects and instrument consumption. Detailed fragmentation analysis found that, under positive ion mode, relative abundance of specific fragment ions m/z 449 to m/z 303 showed linear correlation to the mass content of hesperidin (0% to 100%) with good correlation coefficient (R2 = 0.9958). Utilizing this method, the mass ratio of hesperidin to neohesperidin in Changshanhuyou was relatively quantified as 3.7:96.3 with RSD at 2.9%. Finally, using internal standard method, the absolute quantitative analysis was performed with acceptable reproducibility (RSD 1.3 and 4.5% for intra- and inter-day variations) and recoveries (from 95.9% to 108.9%), acceptable limit of detection (0.33 ng). In general, OLE–QTOF-MS/MS represented a promising and practical method for simple, rapid and effective analysis of isomeric compounds in complex matrices.

Ultra HPLC Method for Fixed Dose Combination of Azilsartan Medoxomil and Chlorthalidone: Identification and in silico Toxicity Prediction of Degradation Products

The fixed dose combination of azilsartan medoxomil potassium and chlorthalidone has been introduced for the effective treatment of hypertension. In the present work a rapid, simple and accurate stability indicating ultra HPLC assay method has been developed. The separation of azilsartan medoxomil, chlorthalidone and their degradation products were accomplished on an Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) column using mobile phase combination of 0.02% trifluoroacetic acid in water and acetonitrile in gradient mode. The forced degradation products were identified using liquid chromatography‒electrospray ionisation-quadrupole time of flight-tandem mass spectrometry (LC‒ESIQTOF–MS/MS) and accurate mass experiments. The in silico toxicities of the degradation products for both the drugs were evaluated. The proposed method was validated as per the ICH Q2 (R1) guideline for selectivity, linearity, precision, accuracy and robustness.

Shufeng Jiedu Capsules Alleviate Lipopolysaccharide-Induced Acute Lung Inflammatory Injury via Activation of GPR18 by Verbenalin

Background/Aims: Acute respiratory tract infection (ARTI) is the most common reason for outpatient physician office visits. Although powerful and significant in the treatment of infections, antibiotics used for ARTI inappropriately have been an important contributor to antibiotic resistance. We previously reported that Shufeng Jiedu Capsule (SJC) can effectively amplify anti-inflammatory signaling during infection. In this study, we aimed to systematically explore its composition and the mechanism of its effects in ARTI. Methods: Pseudomonas aeruginosa (PAK) strain was used to generate a mouse model of ARTI, which were then treated with different drugs or compounds to determine the corresponding anti-inflammatory roles. High-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry. was conducted to detect the chemical compounds in SJC. RNAs from the lung tissues of mice were prepared for microarray analysis to reveal globally altered genes and the pathways involved after SJC treatment. Results: SJC significantly inhibited the expression and secretion of inflammatory factors from PAK-induced mouse lung tissues or lipopolysaccharide-induced peritoneal macrophages. Verbenalin, one of the bioactive compounds identified in SJC, also showed notable anti-inflammatory effects. Microarray data revealed numerous differentially expressed genes among the different treatment groups; here, we focused on studying the role of GPR18. We found that the anti-inflammatory role of verbenalin was attenuated in GPR18 knockout mice compared with wild-type mice, although no statistically significant difference was observed in the untreated PAK-induced mice types. Conclusion: Our data not only showed the chemical composition of SJC, but also demonstrated that verbenalin was a significant anti-inflammatory compound, which may function through GPR18.

Identification of some Bioactive Metabolites in a Fractionated Methanol Extract from Ipomoea aquatica (Aerial Parts) through TLC, HPLC, UPLC-ESI-QTOF-MS and LC-SPE-NMR Fingerprints Analyses.

The plant species Ipomoea aquatica contains various bioactive constituents, e.g. phenols and flavonoids, which have several medical uses. All previous studies were executed in Asia; however, no reports are available from Africa, and the secondary metabolites of this plant species from Africa are still unknown. The present study aims finding suitable conditions to identify the bioactive compounds from different fractions. Chromatographic fingerprint profiles of different fractions were developed using high-performance liquid chromatography (HPLC) and then these conditions were transferred to thin-layer chromatography (TLC). Subsequently, the chemical structure of some bioactive compounds was elucidated using ultra-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry (UPLC-QTOF-MS) and liquid chromatography-solid phase extraction-nuclear magnetic resonance (LC-SPE-NMR) spectroscopy. The HPLC fingerprints, developed on two coupled Chromolith RP-18e columns, using a gradient mobile phase (methanol/water/trifluoroacetic acid, 5:95:0.05, v/v/v), showed more peaks than the TLC profile. The TLC fingerprint allows the identification of the types of chemical constituents, e.g. flavonoids. Two flavonoids (nicotiflorin and ramnazin-3-O-rutinoside) and two phenolic compounds (dihydroxybenzoic acid pentoside and di-pentoside) were tentatively identified by QTOF-MS, while NMR confirmed the structure of rutin and nicotiflorin. The HPLC and TLC results showed that HPLC fingerprints give more and better separated peaks, but TLC helped in determining the class of the active compounds in some fractions. Bioactive constituents were identified as well using MS and NMR analyses. Two flavonoids and two phenolic compounds were tentatively identified in this species for the first time, to the best of our knowledge. Copyright © 2017 John Wiley & Sons, Ltd.

Phenolic compounds responding to zinc and/or cadmium treatments in Gynura pseudochina (L.) DC. extracts and biomass

Gynura pseudochina (L.) DC. is a Zn/Cd hyperaccumulative plant. In an in vivo system under controlled plant age, this research reveals that phenolic compounds and lignification play beneficial roles in protecting G. pseudochina from exposure to an excess of Zn and/or Cd, and Zn reduces Cd toxicity under the dual treatments. The total phenolic content (TPC), total flavonoid content (TFC), and half-maximal inhibitory concentration (IC50) values correspond to the metal dose-response curves. Liquid chromatography-electrospray ionization-quadrupole time of flight-tandem mass spectrometry (LC-ESI-QTOF-MS/MS) is used to characterize phenolic compounds and their glycosides, which could play roles in antioxidant activities and in the esterification of the cell wall, especially derivatives of p-coumaric and caffeic acid. Confocal laser scanning microscopy (CLSM) and micro X-ray fluorescence (μ-XRF) imaging revealed that the accumulation of Zn and Cd in the cell wall involves flavonoid compounds. Low extractable pools of Cd and Zn in the leaf extracts indicate that these elements are tightly bound to the plant biomass structures. The bulk X-ray absorption near edge structure (XANES) spectra indicate that Zn2+ and Cd2+ dominate with O and S ligands, which could be provided by cell walls, phenolic compounds, and sulphur protein. Consequently, the benefit of these results is to support the growth of G. pseudochina for phytoremediation in a Zn- and/or Cd-contaminated site.

Uncommon Modifications and Dehydration Reaction in Analysis of Lysozyme by Matrix-Assisted Laser Desorption Ionization Tandem Time of-Flight Mass Spectrometry

Abstract Tryptic digest of Lysozyme were analyzed by matrix-assisted laser desorption ionization tandem time of flight-mass spectrometry (MALDI-TOF/TOF-MS). Six unique peptides were identified with high confidence by database searching and the Mascot score is 420 with the protein coverage of 54%. Furthermore, several uncommon modifications including deamidation of Asn residue, deamidation of Asn plus oxidation of Met, deamidation of Asn plus oxidation of Met and oxidation of Trp were observed for peptide IVSDGNGMNAWVAWR (98→112). Furthermore, a dehydration reaction was observed in the process of MALDI for this peptide. All above results showed that the Asparagine at 103 rd position has a highly chemical reactivity. In addition, the modification of propionamide (Cystine) was observed for another two peptides. This study indicated that, for those of unmatched data by database searching, the data utilization and the certainty of analysis result would be increased by using manual explanation and selecting very low abundant ions in primary spectrum. This method is also helpful for finding potential post-translational modifications.

Identification Glutamate Methylation by Matrix-assisted Laser Desorption Ionization Tandem Time-of Flight Mass Spectrometry

The separated intracellular proteinase inhibitor and bacillopeptidase F of Bacillus subtilis subsp.were identified by MALDI-TOF/TOF mass spectrometry with high confidence.Furthermore,a total of five peptides concerning methylation of glutamic acid were found by a protein database search and manual explanation,specially,two individual methylation sites of glutamic acid were detected for peptide FELVVYDSEHK.Our results indicated that MALDI-TOF/TOF high energy CID played an important role in the identification of glutamate methylation because it provided abundant fragment information and the full range mass scanning,which improved the certainty of analytical results.In addition,we found the five methylated peptides were always detected simultaneously with their non-methylated analogue and with lower abundance,which indicated that the methylated protein was only a small portion of the total protein.This fact can be used to judge the false positive identification of peptide methylation.Finally,the immonium ion of methylated glutamate at m/z 116 also could provide a clue for this modification compared with the immonium related ion of methylated lysine at m/z 98 and m/z 143.