Exactive Orbitrap Mass Spectrometry Research Articles

Regulatory network analysis reveals gene-metabolite relationships in pear fruit treated with methyl jasmonate

The economic value of pear is determined by its intrinsic qualities, which are influenced by metabolites produced during the ripening process. Methyl jasmonate (MeJA), a hormone, plays an important role in plant metabolism. To date, few studies have investigated the molecular mechanism underlying the changes in metabolic pathways related to the internal quality of pear fruit after MeJA treatment. In this study, ultrahigh-performance liquid chromatography‒Q Exactive Orbitrap mass spectrometry (UHPLC‒QE‒MS) was used to determine the changes in metabolite contents in pear after MeJA treatment. MeJA treatment primarily activated carbohydrate metabolism and amino acid metabolism pathways. Through combined analysis of UHPLC‒QE‒MS data and whole-transcriptome data, the abovementioned pathways and each metabolite were analysed separately, and competitive endogenous RNA (ceRNA) and microRNA–transcription factor–target (miRNA–TF–target) regulatory networks were constructed. The core nodes of three genes (PEA, Pbr022732.1; GAA, Pbr035655.1; and miR8033-x) and two genes (SDS, Pbr031708.1; and novel-m6796-3p) were associated with the carbohydrate metabolism and amino acid metabolism pathways, respectively. The core mRNA nodes TCONS_00048038 and Pbr019584.1, the core miRNA node miR4993-x, the core lncRNA node TCONS_0004356, the core circRNA node novel_circ_001967 and the core transcription factor node TSO1 (Pbr025407.1) were identified via separate metabolite analyses. These findings elucidate the changes in metabolites related to fruit quality in ‘Nanguo’ pear and the relationships between the metabolites and genes, reveal the molecular mechanism underlying the response of MeJA treatment in pear fruit, and provide a theoretical basis for improving the internal quality of ‘Nanguo’ pear.

Persistence and pathway of glyphosate degradation in the coastal wetland soil of central Delaware

Glyphosate is a globally dominant herbicide. Here, we studied the degradation and microbial response to glyphosate application in a wetland soil in central Delaware for controlling invasive species (Phragmites australis). We applied a two-step solid-phase extraction method using molecularly imprinted polymers designed for the separation and enrichment of glyphosate and aminomethylphosphonic acid (AMPA) from soils before their analysis by ultra-high-performance liquid chromatography (UHPLC) and Q Exactive Orbitrap mass spectrometry methods. Our results showed that approximately 90 % of glyphosate degraded over 100 d after application, with AMPA being a minor (<10 %) product. Analysis of glyphosate-specific microbial genes to identify microbial response and function revealed that the expression of the phnJ gene, which codes C-P lyase enzyme, was consistently dominant over the gox gene, which codes glyphosate oxidoreductase enzyme, after glyphosate application. Both gene and concentration data independently suggested that C-P bond cleavage—which forms sarcosine or glycine—was the dominant degradation pathway. This is significant because AMPA, a more toxic product, is reported to be the preferred pathway of glyphosate degradation in other soil and natural environments. The degradation through a safer pathway is encouraging for minimizing the detrimental impacts of glyphosate on the environment.

Detection of sildenafil and its 9 metabolites in a post-race horse urine sample: A case report

The use of prohibited substances in horse racing is a major concern that jeopardizes both the fairness of competitions and the health of horses. This problem can stem from the use of licensed drugs for animal health, as well as unlicensed substances. Horse doping laboratories monitor the potential use of these substances in racehorses within the framework of regulations set by the International Federation of Horse Racing Authority. In this context, sildenafil and its major metabolite n-desmethyl sildenafil were detected in a post-race horse urine sample sent to the Pendik Veterinary Control Institute Doping Control Laboratory through a screening analysis performed with Liquid Chromatography Triple Quadrupole Mass Spectrometry. These results were confirmed by Q Exactive Orbitrap Mass Spectrometry and follow-up analyses were performed. As a result of these analyses; simultaneous detection of 9 metabolites in horse urine was reported, two of them for the first time. In addition, the pioneer and comprehensive data resulting from this study provide preliminary data for future studies and anti-doping analyses.

Targeted quantification and untargeted exploration of furan and derivatives in infant food by headspace extraction-gas chromatography-Q Exactive Orbitrap mass spectrometry

The aim of the present study was to assess the performance and complementarity of methods capable of both quantifying furan, 2-Methylfuran (2-MF) and 3-Methylfuran (3-MF) in infant foods, but also to comprehensively explore other furan derivatives. It is more particularly a question of validating and comparing the couplings of the two headspace extraction methods most used for the analysis of furan compounds − Headspace Solid Phase Microextraction (HS-SPME) and Static HeadSpace (SHS) – with gas chromatography hyphenated to a high-resolution mass detector (Q Exactive-Orbitrap MS) which allows both targeted quantification and suspect screening. Firstly, the accuracy profile approach was implemented to assess, validate and compare HS-SPME- and SHS-GC-Q Exactive-Orbitrap MS for the quantification of furan in two model infant foods, apple puree and first infant formula. SHS-GC-Q Exactive-Orbitrap MS, showed better accuracy (uncertainty < 17.2 % vs 22.5 % for HS-SPME GC-Q Exactive-Orbitrap MS) and better sensitivity (LOQ < 2.8 vs LOQ < 4.0 µg/kg) over a broader validation range (2–100 µg/kg vs 5–100 µg/kg in apple puree). Secondly, SHS-GC-Q Exactive-Orbitrap MS was assessed and validated by accuracy profile for the quantification of 2-MF and 3-MF, with performance close to those for furan except for 3-MF in apple puree. Thirdly, SHS-GC-Q Exactive-Orbitrap MS was used to quantify the levels of these compounds in 20 commercial samples (n = 3) belonging to the four main categories of infant food (infant formulae, fruit purees, infant cereals, vegetable/fish baby meals). Furan was quantified in 75 % of the samples, with maximum levels in the vegetable/fish-based infant foods (up to 127 µg/kg) while 2-MF and 3-MF were quantified in 45 % and 15 % of products respectively, with maximum levels of 14.1 µg/kg in follow-on formula 3rd age and 9.2 µg/kg in apple puree. Finally, SHS- and HS-SPME-GC-Q Exactive-Orbitrap MS data of the 20 infant products were processed in suspect screening mode using Compound DiscovererTM software. Coupling with HS-SPME, it made it possible to identify 13 additional furan derivatives, i.e. 5 more than with SHS. The relevance and safety status of the compounds identified are discussed.

Enzymatic Preparation, In-Depth Molecular Analysis, and In Vitro Digestion Simulation of Palmitoleic Acid (ω-7)-Enriched Fish Oil Triacylglycerols.

In this study, an enzymatic reaction was developed for synthesizing pure triacylglycerols (TAG) with a high content of palmitoleic acid (POA) using fish byproduct oil. The characteristics of synthesized structural TAGs rich in POA (POA-TAG) were analyzed in detail through ultrahigh-performance liquid chromatography Q Exactive orbitrap mass spectrometry. Optimal conditions were thoroughly investigated and determined for reaction systems, including the use of Lipozyme TL IM and Novozym 435, 15 wt % lipase loading, substrate mass ratio of 1:3, and water content of 2.5 and 0.5 wt %, respectively, resulting in yields of 67.50 and 67.45% for POA-TAG, respectively. Multivariate statistical analysis revealed that TAG 16:1/16:1/20:4, TAG 16:1/16:1/16:1, TAG 16:1/16:1/18:1, and TAG 16:0/16:1/18:1 were the main variables in Lipozyme TL IM and Novozym 435 enzyme-catalyzed products under different water content conditions. Finally, the fate of POA-TAG across the gastrointestinal tract was simulated using an in vitro digestion model. The results showed that the maximum release of free fatty acids and apparent rate constants were 71.44% and 0.0347 s-1, respectively, for POA-TAG lipids, and the physical and structural characteristics during digestion depended on their microenvironments. These findings provide a theoretical basis for studying the rational design of POA-structural lipids and exploring the nutritional and functional benefits of POA products.

Integrated serum pharmacochemistry and investigation of the anti-influenza A virus pneumonia effect of Qingjin Huatan decoction

Ethnopharmacological relevanceQingjin Huatan Decoction (QJHTT) consists of 11 herbal medicines: Scutellaria baicalensis Georgi, Gardenia jasminoides J. Ellis, Platycodon grandiflorus (Jacq.) A. DC., Ophiopogon japonicus (Thunb.) Ker Gawl., Morus alba L., Fritillaria thunbergii Miq., Anemarrhena asphodeloides Bunge, Trichosanthes kirilowii Maxim., Citrus reticulata Blanco, Poria cocos (Schw.) Wolf, and Glycyrrhiza uralensis Fisch. As a traditional Chinese medicinal formula, QJHTT has been used for more than 400 years in China. It has shown promising results in treating influenza A virus (IAV) pneumonia. Aim of the studyTo elusive the specific pharmacological constituents and mechanisms underlying its anti-IAV pneumonia effects. Materials and methodsThe components in QJHTT were analyzed through the use of a serum pharmacology-based ultra high-performance liquid chromatography Q- Exactive Orbitrap mass spectrometry (UHPLC-Q Exactive Orbitrap-MS) method. Simultaneously, the dynamic changes in IAV-infected mouse lung viral load, lung index, and expression of lung inflammation factors were monitored by qRT-PCR. ResultsWe successfully identified 152 chemical components within QJHTT, along with 59 absorbed chemical prototype constituents found in the serum of mice treated with QJHTT. 43.45% of these chemical components and 43.10% of the prototype constituents were derived from the monarch drugs, namely Huangqin and Zhizi, aligning perfectly with traditional Chinese medicine theory. Notably, our analysis led to the discovery of 14 compounds within QJHTT for the first time, three of which were absorbed into the bloodstream. Simultaneously, we observed that QJHTT not only reduced the viral load but also modulated the expression of inflammation factors in the lung tissue including TNF-α, IL-1β, IL-4, IL-6, IFN-γ, and IL17A. A time-effect analysis further revealed that QJHTT intervention effectively suppressed the peak of inflammatory responses, demonstrating a robust anti-IAV pneumonia effect. ConclusionsWe comprehensively analyzed the pharmacological material basis of QJHTT by a highly sensitive and high-resolution UHPLC-Q Exactive Orbitrap-MS method, and demonstrated its efficacy in combating IAV pneumonia by reducing lung viral load and inflammatory factors. This study has significant importance for elucidating the pharmacological basis and pharmacological mechanism of QJHTT in combating IAV pneumonia.

Chemical profiling of Simiao pill and quantification of main effective constituents in it by ultra-high-performance liquid chromatography coupled with Q Exactive Orbitrap and triple quadrupole mass spectrometry.

Simiao pill is one of the most commonly used prescriptions in traditional Chinese medicine for the treatment of hyperuricemia and gout. However, methods based on more accurate and comprehensive qualitative and quantitative analyses of the active ingredients are not yet perfect due to limited methodology. This not only hinders the elucidation of the pharmacological mechanism of Simiao pill, but also its comprehensive clinical development and utilization. In this study, we employed ultra-high-performance liquid chromatography-Q Exactive Orbitrap-mass spectrometry technology to perform rapid analysis and identification of the chemical constituents in Simiao pill. A total of 101 chemical components were identified, including 26 alkaloids, 15 terpenoids, 11 flavonoids, eight steroids, six fatty acids, five limonoids, four saponins, five phenylpropanoids, and 21 other compounds. In addition, we established a new method by high-throughput ultra-high-performance liquid chromatography-Q Exactive Orbitrap-mass spectrometry combined with ultra-high-performance liquid chromatography-triple quadrupole-tandem mass spectrometry technology for quantification of 14 main active ingredients, such as adenosine (1), phellodendrine (2), mangnoflorine (3), β-ecdysterone (4), 25R-inokosterone (5), 25S-inokosterone (6), jatrorrhizine (7), palmatine (8), chikusetsu saponin IVa (9), limonin (10), atractylenolide III (11), atractylenolide I (12), obacunone (13), and atractylenolide II (14) in Simiao pill. This work laid a foundation for further analysis and quality control of effective components in Simiao pill.

The chemical profiling and identification of Abelia macrotera (Graebn. et Buchw.) Rehd. based on UHPLC-Q-Exactive Orbitrap MS and study their antioxidant, anti-tyrosinase and anti-inflammatory activities

Although Abelia macrotera has long been used as the raw material for the special snack “immortal tofu” by Chinese population, the composition and activity of its leaf and stem are rarely studied. Ultrahigh-performance liquid chromatography-quadrupole (UHPLC-Q)-Exactive Orbitrap mass spectrometry and various experiments were used for in vitro study of its chemical composition and antioxidant, antityrosinase, and anti-inflammatory activities. A fast, accurate, and sensitive UHPLC-Q-Exactive Orbitrap mass spectrometry method has been established and effectively used for the determination of the chemical composition of the leaf and stem of A. macrotera. The biological activities of the leaf and stem extracts of the A. macrotera were determined in vitro by antioxidant and anti-inflammatory methods. A total of 142 chemical constituents including 48 flavonoids, 65 organic acids, 12 phenylpropanoids, and 19 others were identified by UHPLC-Q-Exactive Orbitrap mass spectrometry. The results show that the leaf and stem extracts have wide and significant antioxidant capacities, including scavenging effects of DPPH, ABTS and hydroxyl radicals, and the ability to reduce both iron and copper metal ions. Additionally, they showed a strong inhibition of NO production, with a dose-dependent inhibition. To the best of our knowledge, this is the first study to report the identification and content determination of the chemical components of the A. macrotera leaf and stem. These results will be beneficial to further understand its medicinal value.

Identification of characteristic flavor compounds and small molecule metabolites during the ripening process of Nuodeng ham by GC-IMS, GC–MS combined with metabolomics

To investigate effects of metabolites and volatile compounds on the quality of Nuodeng ham, gas chromatography-mass spectrometry (GC–MS), ultra-high performance liquid chromatography-Q exactive orbitrap-mass spectrometry (UHPLC-QE-MS), and gas chromatography-ion transfer spectroscopy (GC-IMS) were used to analyze the differences of free fatty acids, small molecule metabolites and volatile compounds of Nuodeng ham at different ripening stages (the first, second and third year sample). 40 free fatty acids were detected. 757 and 300 metabolites were detected in positive and negative ion modes, respectively. 48 differential metabolites (VIP ≥ 1.5, P < 0.05) might important components affecting flavor differences of Nuodeng ham. Metabolic pathways revealed that fermenting-ripening of ham was associated with 31 metabolic pathways, among, 19 pathways were significant (Impact > 0.01, P < 0.05). 58 volatile compounds were identified, combined with PCA and PLS-DA, 15 flavor markers were screened out. These findings provide a scientific basis for further research on the flavor formation mechanism of Nuodeng ham.

Evaluation of Lipidomics Profile of Quinoa Flour and Changes during Storage Based on Ultra Performance Liquid Chromatography Coupled with Quadrupole Exactive Orbitrap Mass Spectrometry.

Although quinoa is nutritious, its high fat content and lipase activity make it easily oxidized during storage. Meanwhile, quinoa's lipid composition and changes during storage are still unknown. Therefore, we stored fresh quinoa flour at low temperature and low humidity (LL), normal temperature and normal humidity (NN), and high temperature and high humidity (HH) conditions for 120 days to assess its oxidative stability and to monitor the changes in lipid composition. Herein, the contents of fatty acids, the peroxide values, the malondialdehyde values, and the lipase activity in quinoa flour during storage are determined to evaluate its oxidation stability. At LL and NN conditions, the contents of fatty acids, the peroxide values, the malondialdehyde values, and the lipase activity changed slowly. They were 3 (LL) and 5 times (NN), 2.7 (LL) and 4.7 times (NN), 1.4 (LL) and 2.3 times (NN), and 1.5 (LL) and 1.6 times (NN) the initial content at storage up to 120 d. However, with the prolongation of storage time under HH conditions, they all increased significantly to 8, 6.6, 3, and 2 times the original content. Moreover, during the storage of quinoa under LL, NN, and HH conditions for 120 days, we continuously monitored the lipid composition of quinoa grains with UPLC-Q-Exactive Orbitrap MS/MS. We identified a total of 14 subclasses of 229 lipids, including 90 significantly different lipid species. PCA and PLS-DA showed that quinoa lipids in HH conditions changed significantly with prolonged storage; among these, the TG and DG classes were the most susceptible to oxidation, which could distinguish fresh quinoa from oxidized quinoa. Simultaneously, we also found that lipase activity has a significant impact on lipid metabolism through correlation analysis, which also indicates that enzyme inactivation treatment can slow down lipid hydrolysis and oxidation during storage. To explore the mechanism of these changes, we also identified twelve important lipid metabolism pathways during quinoa storage. In conclusion, our study advances knowledge of the storage stability and lipid oxidation mechanisms of quinoa and provides a theoretical basis for setting the shelf life of quinoa.

Quality markers of Dajianzhong decoction based on multicomponent qualitative and quantitative analysis combined with network pharmacology and chemometric analysis.

Dajianzhong decoction (DJZD), a classic famous prescription, has a long history of medicinal application. Modern studies have demonstrated its clinical utility in the treatment of postoperative ileus (POI). But none of the current quality evaluation methods for this compound is associated with efficacy. This study aimed to identify the quality markers (Q-Markers) connected to the treatment of POI in DJZD. Ultra-performance liquid chromatography quadrupole Exactive Orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap-MS) was used to identify the main constituents in DJZD. Based on the qualitative results obtained by fingerprinting, chemical pattern recognition (CPR) was used to analyse the key components affecting the quality and finally to establish the network of the active ingredients in DJZD with POI. A total of 64 chemical components were detected. After fingerprint analysis, 13 common peaks were identified. The fingerprint similarity of 15 batches of samples ranged from 0.860 to 1.000. CPR analysis was able to categorically classify 15 batches of DJZD into two groups. And gingerenone A, methyl-6-gingerdiol, 6-gingerol, and hydroxy-β-sanshool contributed to their grouping. Twelve common components interact with the therapeutic targets for treating POI. In addition, the mechanism of this prescription for treating POI may be related to the jurisdiction of the neurological system, the immunological system, and the inflammatory response. This integrated approach can accurately assess and forecast the quality of DJZD, presume the Q-Markers of DJZD for POI, and lay the foundation for studying the theoretical underpinnings and exploring the mechanism of DJZD in the treatment of POI.

Determination of multi-mycotoxins in vegetable oil via liquid chromatography-high resolution mass spectrometry assisted by a complementary liquid–liquid extraction

The simultaneous determination of multi-mycotoxins in food commodities are highly desirable due to their potential toxic effects and mass consumption of foods. Herein, liquid chromatography-quadrupole exactive orbitrap mass spectrometry was proposed to analyze multi-mycotoxins in commercial vegetable oils. Specifically, the method featured a successive liquid–liquid extraction process, in which the complementary solvents consisted of acetonitrile and water were optimized. Resultantly, matrix effects were reduced greatly. External calibration approach revealed good quantification property for each analyte. Under optimal conditions, the recovery ranging from 80.8% to 109.7%, relative standard deviation less than 11.7%, and good limit of quantification (0.35 to 45.4 ng/g) were achieved. The high accuracy of proposed method was also validated. The detection of 20 commercial vegetable oils revealed that aflatoxins B1 and B2, zearalenone were observed in 10 real samples. The as-developed method is simple and low-cost, which merits the wide applications for scanning mycotoxins in oil matrices.

Metabolite Analysis of Alternaria Mycotoxins by LC-MS/MS and Multiple Tools

Alternaria fungi are widely distributed plant pathogens that invade crop products, causing significant economic damage. In addition, toxic secondary metabolites produced by the fungi can also endanger consumers. Many of these secondary metabolites are chemically characterized as mycotoxins. In this study, Q Exactive Orbitrap mass spectrometry was used for the non-targeted analysis of the metabolome of seven Alternaria isolates cultured on Potato Carrot Agar (PCA), Potato Dextrose Agar (PDA) and Potato Sucrose Agar (PSA) medium. Due to the difficulty of detecting modified toxins, an analytical strategy with multiple visual analysis tools was also used to determine the presence of sulfate conjugated toxins, as well as to visualize the molecular network of Alternaria toxins. The results show that PSA medium exhibits more advantageous properties for the culture of Alternaria, with more toxigenic species and quantities and more obvious metabolic pathways. Based on high-resolution tandem mass spectrometry (MS/MS) data, the mycotoxins and their metabolites were mainly clustered into four groups: alternariol (AOH)/alternariol monomethyl ether (AME)/altenusin (ALU)/altenuene (ALT)/dehydroaltenusin (DHA)/Desmethyldehydroaltenusin (DMDA) families, Altertoxin-I (ATX-I) family, tentoxin (TEN) family and tenuazonic acid (TeA) family. Moreover, the PSA medium is more suitable for the accumulation of AOH, AME, ALU, ALT, DHA and DMDA, while the PDA medium is more suitable for the accumulation of ATX-I, TEN and TeA. This research may provide theoretical support for the metabolomics study of Alternaria.

The Metabolites and Mechanism Analysis of Genistin against Hyperlipidemia via the UHPLC-Q-Exactive Orbitrap Mass Spectrometer and Metabolomics.

Genistin, an isoflavone, has been reported to have multiple activities. However, its improvement of hyperlipidemia is still unclear, and the same is true with regard to its mechanism. In this study, a high-fat diet (HFD) was used to induce a hyperlipidemic rat model. The metabolites of genistin in normal and hyperlipidemic rats were first identified to cause metabolic differences with Ultra-High-Performance Liquid Chromatography Quadrupole Exactive Orbitrap Mass Spectrometry (UHPLC-Q-Exactive Orbitrap MS). The relevant factors were determined via ELISA, and the pathological changes of liver tissue were examined via H&E staining and Oil red O staining, which evaluated the functions of genistin. The related mechanism was elucidated through metabolomics and Spearman correlation analysis. The results showed that 13 metabolites of genistin were identified in plasma from normal and hyperlipidemic rats. Of those metabolites, seven were found in normal rat, and three existed in two models, with those metabolites being involved in the reactions of decarbonylation, arabinosylation, hydroxylation, and methylation. Three metabolites, including the product of dehydroxymethylation, decarbonylation, and carbonyl hydrogenation, were identified in hyperlipidemic rats for the first time. Accordingly, the pharmacodynamic results first revealed that genistin could significantly reduce the level of lipid factors (p < 0.05), inhibited lipid accumulation in the liver, and reversed the liver function abnormalities caused by lipid peroxidation. For metabolomics results, HFD could significantly alter the levels of 15 endogenous metabolites, and genistin could reverse them. Creatine might be a beneficial biomarker for the activity of genistin against hyperlipidemia, as revealed via multivariate correlation analysis. These results, which have not been reported in the previous literature, may provide the foundation for genistin as a new lipid-lowering agent.

Usnea improves high-fat diet- and vitamin D3-induced atherosclerosis in rats by remodeling intestinal flora homeostasis.

Background: Usnea has various pharmacological properties, including anti-inflammatory, antitumor, antioxidant, antiviral, and cardiovasculoprotective effects. Aim of the study: To investigate the potential mechanisms underlying the anti-atherosclerosis (AS) activity of Usnea ethanol extract (UEE) via the regulation of intestinal flora. Materials and Methods: The chemical composition of UEE was determined using ultra-performance liquid chromatography with quadrupole exactive orbitrap mass spectrometry (UPLC-Q-EOMS). Thirty-six male Sprague-Dawley rats were divided into six groups. A high-fat diet and intraperitoneal vitamin D3 injections were used to establish a rat model of AS. After 4weeks of treatment with UEE, hematoxylin-eosin staining was performed to evaluate the pathomorphology of the aorta, liver, and colon. The composition and diversity of the rat intestinal flora were determined using high-throughput 16S rRNA sequencing. Enzyme-linked immunosorbent assays were used to measure the levels of plasma trimethylamine oxide (TMAO), serum bile acid (BA), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). The protein expression of cholesterol 7α-hydroxylase (CYP7A1) and flavin monooxygenase 3 (FMO3) in the liver and zonula occludens-1 (ZO-1) and occludin in colon tissue was detected via western blotting. Results: Forty-four compounds were identified in UEE. In the rat model of AS, UEE significantly prevented calcium deposition; decreased the serum levels of TC, TG, LDL-C, LPS, TNF-α, and IL-6; and increased the serum level of HDL-C. Additionally, all UEE dosages decreased the relative abundance of Verrucomicrobiota while increased that of Bacteroidetes. FMO3 protein expression and TMAO levels decreased, whereas CYP7A1 protein expression and BA levels increased. The absorption of intestinal-derived LPS was minimized. Furthermore, the protein expression of ZO-1 and occludin was upregulated. Conclusion: UEE ameliorated AS. The underlying mechanism was the reversal of imbalances in the intestinal flora by Usnea, thereby inhibiting calcium deposition, abnormal lipid metabolism, and inflammatory response.

Drug metabolite cluster centers-based strategy for comprehensive profiling of Neomangiferin metabolites in vivo and in vitro and network pharmacology study on anti-inflammatory mechanism

Neomangiferin (NMF) is an extremely special xanthone that could be simultaneously attributed to C-glycoside and O-glycoside with a variety of biological activities, such as anti-inflammatory, antitumor, antipyretic, and so on. So far as we know, the metabolism profiling has been insufficient until now. Herein, Drug Metabolite Cluster Centers (DMCCs)-based Strategy has been developed to profile the NMF metabolites in vivo and in vitro. Firstly, the DMCCs was proposed depending on literature-related and preliminary analysis results. Secondly, the specific metabolic rule was implemented to screen the metabolites of candidate DMCCs from the acquired Ultra High Performance Liquid Chromatography Quadrupole Exactive Orbitrap Mass Spectrometry (UHPLC-Q-Exactive Orbitrap MS) data by extracted ion chromatography (EIC) method. Thirdly, candidate metabolites were accurately and tentatively identified according to the pyrolysis law of mass spectrometry, literature reports, comparison of reference substances, and especially the diagnostic product ions (DPIs) deduced preliminarily. Finally, network pharmacology was adopted to elucidate the anti-inflammatory action mechanism of NMF on the basis of DMCCs. As a result, 3 critical metabolites including NMF, Mangiferin (MF) and Norathyriol (NA) were proposed as DMCCs, and a total of 61 NMF metabolites (NMF included) were finally screened and characterized coupled with 3 different biological sample preparation methods including solid phase extraction (SPE), acetonitrile precipitation and methanol precipitation. Among them, 32 metabolites were discovered in rat urine, 30 in rat plasma, 12 in rat liver, 9 metabolites in liver microsomes and 8 in rat faeces, respectively. Our results also illustrated that NMF primarily underwent deglucosylation, glucuronidation, methylation, sulfation, dihydroxylation and their composite reactions in vivo and in vitro. Additionally, network pharmacology analysis based on DMCCs revealed 85 common targets of disease-metabolites, and the key targets were TNF, EGFR, ESR1, PTGS2, HIF1A, IL-2, PRKCA and PRKCB. They exerted anti-inflammatory effects mainly through the pathways of inflammatory response, calcium-dependent protein kinase C activity, nitrogen metabolism, pathways in cancer and so on. In general, our study constructed a novel strategy to comprehensive elucidate the biotransformation pathways of NMF in vivo and in vitro, and provided vital reference for further understanding its anti-inflammatory action mechanism. Moreover, the established strategy could be generalized to the metabolism and action mechanism study of other natural products.

Combining ultra-high-performance liquid chromatography quadruple exactive orbitrap mass spectrometry with chemometrics to identify and verify the blood-activating components of hawthorn.

Hawthorn, one of the widely-used Chinese herbal medicines, has been used to treat blood stasis syndrome in the clinic, but its blood-activating components are unclear. This study combined the ultra-high-performance liquid chromatography-quadruple exactive-orbitrap mass spectrometry with chemometrics to identify the blood-activating components of hawthorn. Different polar fractions of hawthorn aqueous extracts were extracted and mixed to prepare 14 samples. The contents of 25 chemical components for 14 samples were determined by the proposed quantitative method which was validated in terms of linearity, precision, stability, repeatability, and recovery, while the blood-activating effect was evaluated by measuring the whole blood viscosity, plasma viscosity, and plasma fibrinogen levels. Then the partial least squares model was established on the spectrum-effect relationship. The result showed that vitexin-2″-O-rhamnoside, rutin, citric acid, malic acid, gallic acid, and fumaric acid could reduce the whole blood viscosity, plasma viscosity, and plasma fibrinogen levels in blood stasis model rats, and these components were the blood-activating components of hawthorn. This study provided a scientific basis for clarifying the blood-activating components of hawthorn, and the spectrum-effect approach proved to be an effective approach to discovering the bioactive components of Chinese herbal medicines.

High-coverage lipidomics analysis reveals biomarkers for diagnosis of acute exacerbation of chronic obstructive pulmonary disease

Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is the leading cause of morbidity and mortality in COPD management. However, detecting the progression from the stable stage to acute exacerbation mainly depends on doctors’ judgment of clinical symptoms, and there is no biomarker that can be used for auxiliary clinical diagnosis. In this work, serum samples from COPD patients (n = 82) and healthy subjects (n = 29) were collected and analyzed. Patients with COPD were divided into stable COPD (SCOPD) and AECOPD groups, with the latter comprising subtypes 1 and 2. High-coverage lipidomics profiling of 913 lipids belonging to 19 subclasses was carried out by liquid chromatography–Q–Exactive orbitrap mass spectrometry. We performed 4 cross-comparisons to characterize metabolic disturbances associated with the progression of stable COPD to AECOPD—ie, SCOPD vs healthy subjects, AECOPD vs SCOPD, AECOPD subtype 1 vs SCOPD, and AECOPD subtype 2 vs SCOPD. We tentatively identified 86 lipids with differential abundance among groups, lipids that were altered from the stable stage of disease to AECOPD included sphingolipids, ether-containing glycerophospholipids, phosphatidylglycerols, and glycerol lipids. Three panels of lipid biomarkers specific to AECOPD, AECOPD subtypes 1 and 2 vs SCOPD yielded areas under the receiver operating characteristic curve of 0.788, 0.921 and 0.920, respectively, with sensitivity of 77.5%, 80.7% and 91.3%, respectively, and specificity of 75.8%, 97.0% and 87.9%, respectively. The result indicated differences in lipid metabolism may underlie AECOPD and its 2 subtypes and can serve as biomarkers for early diagnosis, and high-coverage lipidomics proved to be an accurate approach to profile the lipid metabolism in biological samples.

The regulating effect of light on the content of flavan-3-ols and derivatives of hydroxybenzoic acids in the callus culture of the tea plant, Camellia sinensis L

The objective was the study of the regulating effect of light on the composition and contents of phenolic compounds in the callus culture of the tea plant (Camellia sinensis) grown for 28 days in the light (16-h photoperiod) or in the dark. For the analyses of phenolic compounds, an ultra-performance liquid chromatography in combination with a photodiode array detector and a high-resolution Q Exactive Orbitrap mass spectrometry (UPLC-PDA-HRMS/MS) was used. Nineteen phenolic compounds were characterized. The main phenolic compounds were flavan-3-ols (mono- and oligomers of flavan-3-ols) and derivatives of hydroxybenzoic acids (mono- and digalloyl glucoses, hexosides of 2,5- and 2,3-dihydroxybenzoic and salicylic acids). It was shown that the growth of the tea callus in the light or in the dark does not affect the composition of phenolic compounds, but differently affects the content of flavan-3-ols and derivatives of hydroxybenzoic acids. In the callus grown in the light, the content of flavan-3-ols was about 2-fold higher than in the dark. On the contrary, the content of derivatives of hydroxybenzoic acids in the light was at average 3-fold lower. The regulating role of light in the biosynthesis of phenolic compounds, as well as light control of the distribution of carbon flux between the branches of the shikimate pathway, which are associated with the biosynthesis of flavan-3-ols and hydroxybenzoic acids, are discussed.

Integrating approach to discover novel bergenin derivatives and phenolics with antioxidant and anti-inflammatory activities from bio-active fraction of Syzygium brachythyrsum

Syzygium brachythyrsum is an important folk medicinal and edible plant in Yunnan ethnic minority community of China, however, little is known about the chemical and bio-active properties. The present study is aimed to identify the bioactive constituents with antioxidant and anti-inflammatory properties by an integrating approach. First, two new bergenin derivatives, brachythol A (1) and brachythol B (2) , together with eleven known phenolic compounds (3–13) were isolated from bioactive fractions by phytochemical method. Among these isolated chemicals, five bergenin derivatives, along with 3 phenolics were found in Syzygium genus for the first time. Then, a further chemical investigation based on ultra-high-performance liquid chromatography-Q Exactive Orbitrap mass spectrometry resulted in a total of 107 compounds characterized in the bio-active fractions, including 50 bergenin derivatives, among which 14 bergenin derivatives and 14 phenolics were potential new natural chemicals. Most of the isolated compounds showed obvious antioxidant activities, while compounds 11, 12, and 13 had favorable performance. Eight compounds (2–5, 7, and 9–11) showed good inhibitory activity on nitric oxide (NO) production in macrophage RAW 264.7 cells. The structure–activity correlation analysis indicated that the antioxidation and anti-inflammatory activities enhanced when bergenin was esterified with gallic acid, caffeic acid or ferulic acid. This is the first report of bergenins in Syzygium genus and the richness in new bio-active bergenins and gallic acid derivatives indicated that Syzygium brachythyrsum is a promising functional and medicinal resource.