Flight Tandem Mass Spectrometry Research Articles

Metabolite identification of salvianolic acid A in rat using post collision-induced dissociation energy-resolved mass spectrometry

BackgroundAs one of the most famous natural products, salvianolic acid A (SAA) is undergoing clinical trials for the treatments of angina pectoris and coronary heart disorders. However, the in vivo metabolites of SAA have only been tentatively identified, leading to a barrier for precise therapeutical drug monitoring.MethodsUltra-high performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry (UPLC–Qtof-MS/MS) was firstly employed to acquire high-resolution MS1 and MS2 spectra for all metabolites. Through paying special attention onto the features of ester bond dissociation, metabolism sites were restricted at certain regions. To further determine the metabolism site, such as the monomethylated products (M23, M25, and M26), post collision-induced dissociation energy-resolved mass spectrometry (post-CID ER-MS) was proposed through programming progressive exciting energies to the second collision chamber of hybrid triple quadrupole-linear ion trap mass spectrometry (Qtrap-MS) device.ResultsAfter SAA oral administration, 29 metabolites (M1–M29), including five, thirteen, and sixteen ones in rat plasma, urine, and feces, respectively, were detected in rats. The metabolism route was initially determined by applying well-defined mass fragmentation pathways to those HR-m/z values of precursor and fragment ions. Metabolism site was limited to SAF- or DSS-unit based on the fragmentation patterns of ester functional group. Through matching the dissociation trajectories of concerned 1st-generation fragment ions with expected decomposition product anions using post-CID ER-MS strategy, M23 and M25 were unequivocally assigned as 3'-methyl-SAA and 3''-methyl-SAA, and M26 was identified as 2-methyl-SAA or 3-methyl-SAA. Hydrolysis, methylation, glucuronidation, sulfation, and oxidation were the primary metabolism channels being responsible for the metabolites' generation.ConclusionTogether, the metabolism regions and sites of SAA metabolites were sequentially identified based on the ester bond dissociation features and post-CID ER-MS strategy. Importantly, the present study provided a promising way to elevate the structural identification confidence of natural products and metabolites.Graphical abstract

Identification and structural characterization of potential degradation products of baricitinib using liquid chromatography combined with quadrupole time-of-flight mass spectrometry

AbstractBaricitinib (BRT) is a drug substance with potent anti-inflammatory activity indicated in rheumatoid arthritis, atopic dermatitis, severe alopecia areata and recently for the treatment of Covid-19. Process impurities of the drug during its formulation are quite known, however studies regarding its degradation products (DPs) under stress conditions are limited. In this study, the drug was subjected to forced degradation under various degradation conditions, including acidic hydrolysis, alkaline hydrolysis, oxidative and thermal, to determine its inherent stability. To this purpose, a novel HPLC method was developed for the determination of degradation impurities of BRT. Alkaline hydrolysis test showed a selectivity towards breaking C–C bonds. This resulted to five DPs formed by chain scission reactions occurred at the pyrrolo-pyrimidine group between C6–C10 and C8–C9. Also, the ethylsulfonyl-azetidin-ylidene group was subjected to C–C bond cleavage at C12–C15 and C16–C18. Degradation products were further characterized with the use of liquid chromatography quadrupole time of flight tandem mass spectrometry (LC-Q-TOF-MS/MS).

Unraveling the Mechanism of Xiaochaihu Granules in Alleviating Yeast-Induced Fever Based on Network Analysis and Experimental Validation.

Xiaochaihu granules (XCHG) are extensively used to treat fever. Nevertheless, the underlying mechanism remains elusive. This study aimed to explore the potential of XCHG in mitigating yeast-induced fever and the underlying metabolic pathways. The chemical composition of XCHG was ascertained using ultra-fast liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (UFLC-Q-TOF-MS/MS), followed by integrated network analysis to predict potential targets. We then conducted experimental validation using pharmacological assays and metabolomics analysis in a yeast-induced mouse fever model. The study identified 133 compounds in XCHG, resulting in the development of a comprehensive network of herb-compound-biological functional modules. Subsequently, molecular dynamic (MD) simulations confirmed the stability of the complexes, including γ-aminobutyric acid B receptor 2 (GABBR2)-saikosaponin C, prostaglandin endoperoxide synthases (PTGS2)-lobetyolin, and NF-κB inhibitor IκBα (NFKBIA)-glycyrrhizic acid. Animal experiments demonstrated that XCHG reduced yeast-induced elevation in NFKBIA's downstream regulators [interleukin (IL)-1β and IL-8], inhibited PTGS2 activity, and consequently decreased prostaglandin E2 (PGE2) levels. XCHG also downregulated the levels of 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA), corticotropin releasing hormone (CRH), and adrenocorticotrophin (ACTH). These corroborated the network analysis results indicating XCHG's effectiveness against fever in targeting NFKBIA, PTGS2, and GABBR2. The hypothalamus metabolomics analysis identified 14 distinct metabolites as potential antipyretic biomarkers of XCHG. In conclusion, our findings suggest that XCHG alleviates yeast-induced fever by regulating inflammation/immune responses, neuromodulation, and metabolism modules, providing a scientific basis for the anti-inflammatory and antipyretic properties of XCHG.

Phytochemical exploration of Neolitsea pallens leaves using UPLC-Q-TOF-MS/MS approach

Neolitsea pallens (D. Don) Momiyama & H. Hara (Family: Lauraceae), commonly known as Pale Litsea, is an evergreen small tree, distributed in India at altitudes of 1500–3000 m. Traditionally utilized for various purposes, its leaves and bark are used as spices, and the plant is valued in preparing a hair tonic from freshly pressed juice. Secondary metabolites of the leaves have not comprehensively been analysed so far. The objective of the study was to determine the chemical composition of the leaves by analysing their 25% aqueous methanol extract with the aid of ultra-performance liquid chromatography quadrupole time of flight tandem mass spectrometry. Overall, 56 compounds were identified in the study. Phenolics represented by phenolic acids, phenolic glycosides, proanthocyanidins, and flavonoids were the main components of the extract.

Homogeneous oxidation of EE2 by ozone: Influencing factors, degradation pathway, and toxicity assessment

17α-Ethinylestradiol(EE2) is a common hormone drug that plays an essential role in endocrine and reproductive health. However, estrogens are degraded only to a limited extent by humans and livestock themselves, and most of them are excreted in undegraded or partially degraded form from human and livestock feces and urine into water bodies, posing a austere ecological risk. Of the known degradation processes in wastewater treatment plants, ozone-induced advanced oxidation shows tremendous potential. At the same time, the limitations of the test methodology makes it extremely difficult to do an in-depth investigation of the degradation mechanism. In this study, the influencing factors, including ozone dose, initial concentration, and pH were investigated. The degradation pathways of EE2 were analyzed by combing with quantum chemical calculations and high performance liquid chromatography coupled with quadrupole-time of flight tandem mass spectrometry (HPLC-Q-TOF-MS). Toxicity Estimation Software Tool (T.E.S.T) evaluated EE2 and its degradation intermediates for a variety of toxicities. And the results showed that reactions such as oxidative hydroxylation of the benzene ring and oxidative ring opening of the aliphatic ring mainly occurred during the degradation of estrogens, and the reaction was initially fast. The degradation effect is lower under neutral conditions, and the acidity and alkalinity of the solution are reduced with the reaction process under strong acid and alkaline conditions. The products of the experimental reactions reflect the active site, e.g., product P1 reflects C(5) as the site of easy reaction, which is consistent with previous theoretical predictions, suggesting that theoretical studies can provide complementary information on oxidation. The toxicity of intermediates obtained from partial ozone degradation improved compared to EE2, and the mutagenicity of EE2 was elevated greatly. However, single ozonation significantly diminished the developmental toxicity and bioaccumulation factor for a large proportion of intermediates. This work may contribute to the theoretical foundation for the practical application of ozone oxidation of environmental estrogens.

Ultra-high-performance liquid chromatography-quadrupole time of flight tandem mass spectrometry based in vitro metabolite profiling of DK-GV-04P, a novel anticancer molecule under drug discovery.

DK-GV-04P, chemically identified as 3-cinnamyl-2-(4-methoxyphenyl) quinazolin-4(3H)-one, is an investigational molecule synthesized at the Chemical Biology Laboratory of the National Institute of Pharmaceutical Education and Research-Ahmedabad. The compound has shown potential anticancer activity against squamous CAL27 cell lines. Metabolite identification and characterization are critical in drug discovery, providing key insights into a compound's pharmacokinetics, pharmacodynamics safety, and metabolic fate. The primary aim of the study was to identify and characterize the in vitro metabolites of DK-GV-04P. In silico identification of the site of metabolism was also carried out using xenosite online software. The molecule was incubated with human liver microsomes and human S9 liver fraction to generate in vitro metabolites, which were further identified and characterized using ultra-high-performance liquid chromatography-quadrupole time of flight tandem mass spectrometry. A total of nine metabolites (four phase I and five phase II) were identified and characterized through tandem mass spectrometry. The major biotransformation pathways involved in metabolism of DK-GV-04P were hydroxylation, O-demethylation and glucuronidation. In addition to this, a detailed biotransformation pathway of DK-GV-04P has been established in this study.

Rapid and comprehensive metabolites identification of 5-demethylnobiletin in rats using UPLC/Triple-TOF-MS/MS based on multiple mass defect filter and their neuroprotection against ferroptosis

5-Demethylnobiletin (5-deNOB) is a hydroxylated polymethoxyflavone (PMF) from Citrus plants known for its neurotrophic, anti-tumor, and antioxidant bioactivities. An ultra-high performance liquid chromatography coupled with triple-time of flight tandem mass spectrometry (UPLC/Triple-TOF-MS/MS) analysis combining with multiple mass defect filter (MMDF) and MetabolitePilot™ was employed to detect and characterize the metabolites of 5-deNOB in rats. A total of 130 metabolites were identified in rats, with 100, 25, 34, and 52 metabolites found in urine, plasma, bile, and feces, respectively. The major metabolic pathways involved demethylation, hydroxylation, dehydroxylation, glucuronidation, and methylation. In a bioassay of evaluating neuroprotection against ferroptosis in PC12 cells, most of the metabolites exhibited superior activity compared to 5-deNOB. These results provide valuable insights into the in vivo pharmacodynamic properties of 5-deNOB and offer potential active small molecules for neuroprotective therapy. Furthermore, the findings demonstrate the effectiveness of UPLC/Triple-TOF-MS/MS combined with MMDF and MetabolitePilot™ for rapid discovery and identification of the in vivo metabolites of natural products.

An antiviral optimized extract from Sanguisorba officinalis L. roots using response surface methodology, and its efficacy in controlling viral hemorrhagic septicemia of olive flounder (Paralichthys olivaceus)

Viral hemorrhagic septicemia causes considerable economic losses for Korea's olive flounder (Paralichthys olivaceus) aquaculture farms; therefore, effective antiviral agents for controlling viral hemorrhagic septicemia virus (VHSV) infection are imperative. The present study implemented a Box–Behnken design and cytopathic reduction assay to derive an optimized extract of Sanguisorba officinalis L. roots (OE-SOR) with maximum antiviral activity against VHSV. OE-SOR prepared under optimized extraction conditions (55% ethanol concentration at 50 °C for 5 h) exhibited potent antiviral activity against VHSV, with a 50% effective 0.21 μg/mL concentration and a 340 selective index. OE-SOR also showed direct virucidal activity in the plaque reduction assay. Administering OE-SOR to olive flounder exhibited substantial efficacies against VHSV infection. Fish receiving 100 mg/kg body weight/day of OE-SOR as a preventive (40.0%; p < 0.05) or therapeutic (44.4%; p < 0.05) exhibited a higher relative survival than the untreated VHSV-infected control group (mortalities of 100% and 90%, respectively). In addition, fish fed with OE-SOR (100 mg/kg body weight/day) for two weeks conveyed a significantly higher inflammatory cytokine expression (nuclear factor kappa-light-chain-enhancer of activated B cells [NF-κB], interleukin-1 beta [IL-1β], and tumor necrosis factor-alpha [TNF-α]) than the control group one to two days post-administration.Moreover, no hematological or histological changes were observed in olive flounder treated with OE-SOR over four weeks. Liquid chromatography-quadrupole-time of flight tandem mass spectrometry and -triple quadrupole tandem mass spectrometry analyses identified ziyuglycoside I as a prominent OE-SOR constituent and marker compound (content: 14.5%). This study verifies that OE-SOR is an effective alternative for controlling viral hemorrhagic septicemia in olive flounder farms as it exhibits efficient in vivo anti-VHSV activity and increases innate immune responses.

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.

Mass Spectrometric Analysis of Cucurbitacins and Dihydrocucurbitacins from the Tuber of Citrullus naudinianus.

The vast pool of structurally and functionally distinct secondary metabolites (i.e., natural products (NPs)) is constantly being expanded, a process also driven by the rapid progress in the development of analytical techniques. Such NPs often show potent biological activities and are therefore prime candidates for drug development and medical applications. The ethyl acetate extract of the tuber of Citrullus naudinianus (C. naudinianus), an African melon with edible fruits and seeds, shows in vitro immunomodulatory activity presumably elicited by cucurbitacins that are known major constituents of this plant. Further potentially immunomodulatory cucurbitacins or cucurbitacin derivatives were assumed to be in the tuber. Given the typically high content of cucurbitacins with similar physicochemical features but often distinct bioactivities, an efficient and reliable separation process is a prerequisite for their detailed characterization and assessment in terms of bioactivity. We therefore developed a detection method to screen and differentiate cucurbitacins via high-performance liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (HPLC-QTOF-MS/MS). In order to confirm the identification, the fragmentation patterns of two cucurbitacins and one 23,24-dihydrocucurbitacin were also investigated. Six characteristic fragments were identified and three of them were employed for the identification of cucurbitacins and 23,24-dihydrocucurbitacins in the extract. As a result, in addition to eight previously reported cucurbitacins from this plant four distinct 23,24-dihydrocucurbitacins (B, D, E, and I) were putatively identified and newly found in the ethyl acetate extract of the tuber of C. naudinianus. The established methodology enables rapid and efficient LC-MS-based analysis and identification of cucurbitacins and 23,24-dihydrocucurbitacins in plant extracts.

Screening, identification and control of unknown estrogen-like compounds in Maillard reaction products of glucose-arginine/lysine model systems

It was speculated that estrogen-like compounds may be produced by chemical reactions during food processing, such as Maillard reaction, which would disrupt the endocrine system of organisms. Herein, the Maillard reaction in the process of high temperature for long time was simulated by using model system, and unknown estrogen-like compounds produced during Maillard reaction were screened by colorimetric assay based on dual estrogen receptor (ER)-gold nanoparticles (AuNPs) and enzyme-linked immunosorbent assay (ELISA). Possible structures of estrogen-like compounds were inferred by ultra-performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (UPLC-QTOF/MS) in combination with a mass database, and finally the structure of estrogen-like compound, 2, 4-dihydroxy-1, 4-benzoxazin-3-one-2-o-β-D-glucopyranoside (DIBOA-glc), was identified by high resolution orbitrap mass spectrometry (Orbitrap HRMS). This is the first study of the screening and identification of unknown estrogen-like compounds produced in Maillard reaction. Additionally, strategy of controlling the formation of DIBOA-glc by adding vitamin B6 in Maillard reaction was proposed, providing effective proposals for the safety control in actual food processing.

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.

Chemical compositions of Souliea vaginata (Maxim) Franch rhizome and their potential therapeutic effects on collagen-induced arthritis in rats.

Rheumatoid arthritis (RA) is a global prevalent chronic autoimmune inflammatory disease and acceptable safety drugs are lack for its treatment. The rhizomes of Souliea vaginata (Maxim) Franch (SV) possess anti-inflammatory functions and are used as substitution of Coptis chinensis Franch. SV is also traditional Chinese medicine and Tibetan medicine for the treatment of conjunctivitis, enteritis and rheumatic. For searching complementary and alternative anti-RA drugs, it is necessary to characterize the potential anti-arthritic activity of SV and underlying mechanism involved. The aim of the study was to test the chemical compositions, evaluate the anti-arthritic effects and underlying mechanisms of SV. The chemical compositions of SV were analyzed using liquid chromatography-ion trap-time of flight tandem mass spectrometry (LCMS-IT-TOF). From day 11 to day 31, SV (0.5, 1.0 and 1.5 g/kg body weight) and Tripterygium glycosidorum (TG, 10 mg/kg body weight) were administered orally to the CIA model rats once a day. Thickness of paw and body weights were measured once every two days from day 1 to day 31. Histopathological changes were measured using hematoxylin-eosin (HE) staining. Effects of SV on the levels of IL-2, TNF-α, IFN-γ, IL-4 and IL-10 in serum of CIA rats were measured by enzyme-linked immunosorbent assay (ELISA) kits. CD3+, CD4+, CD8+ and CD4+CD25+ T cells populations were measured using flow cytometric analysis. To evaluate the possible hepatotoxicity and nephrotoxicity, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea (UREA) and creatinine (CREA) in CIA rats were also tested using blood auto analyzer. 34 compounds were identified from SV based on LCMS-IT-TOF, and triterpenoids are major anti-arthritic compositions. SV significantly relieved CIA rats' paw swelling without obvious influence on the body weight growth. SV decreased the serum levels of IL-2, TNF-α and IFN-γ in CIA rat, and increased the serum levels of IL-4 and IL-10. SV significantly increased and decreased the percentages of CD4+ and CD8+, with no significant effects on CD3+ in lymphocytes of CIA model rats. Moreover, SV simultaneously decreased thymus and spleen indexes and no hepatotoxicity and nephrotoxicity was observed after short-term treatment. These findings suggest that SV possesses preventive and therapeutic effect on RA by modulating the inflammatory cytokines, T-lymphocyte, thymus and spleen indexes and shows no hepatotoxicity and nephrotoxicity.

Protective effects of Liupao tea against high-fat diet/cold exposure-induced irritable bowel syndrome in rats

Liupao tea as a type of dark tea can relieve irritable bowel syndrome by regulating gut microbiota, but the mechanism has not been fully explained. An ultra-high performance liquid chromatography along with quadrupole time of flight tandem mass spectrometry was used to analyze the phytochemicals in Liupao tea. Then, we explored the effects of Liupao tea against IBS. From the results of chemical analysis, we identified catechins, polyphenols, amino acids, caffeine, polysaccharides and other components in Liupao tea. The open-field test, gastrointestinal function-related indexes, histochemical assays, measurements of cytokine and aquaporin 3 (AQP3), and determination of serum metabolites were utilized to monitor the physiological consequences of Liupao tea administration in rats with irritable bowel syndrome. The results showed that Liupao tea had a significant protective effect on irritable bowel syndrome. Liupao tea increased locomotive velocity while reducing interleukin-6, interleukin-1β, and tumor necrosis factor-α levels, as well as gastrointestinal injury. Moreover, Liupao tea increased the AQP3 levels of renal tissues but reduced the AQP3 levels of gastrointestinal tissues. Liupao tea reduced the Firmicutes/Bacteroides ratio and significantly reconstructed the microbial pattern. Liupao tea relieved irritable bowel syndrome by repairing gastrointestinal dysfunction, regulating the secretion of pro-inflammatory cytokines, modulating water metabolism, and restoring microbial homeostasis.

Identification of novel serum autoantibody biomarkers for early esophageal squamous cell carcinoma and high-grade intraepithelial neoplasia detection.

Early diagnosis of esophageal squamous cell carcinoma (ESCC) is critical for effective treatment and optimal prognosis; however, less study on serum biomarkers for the early ESCC detection has been reported. The aim of this study was to identify and evaluate several serum autoantibody biomarkers in early ESCC. We initially screened candidate tumor-associated autoantibodies (TAAbs) associated with ESCC by serological proteome analysis (SERPA) combined with nanoliter-liquid chromatography combined with quadrupole time of flight tandem mass spectrometry (nano-LC-Q-TOF-MS/MS), and the TAAbs were further subjected to analysis by Enzyme-linked immunosorbent assay (ELISA) in a clinical cohort (386 participants, including 161 patients with ESCC, 49 patients with high-grade intraepithelial neoplasia [HGIN] and 176 healthy controls [HC]). Receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic performance. The serum levels of CETN2 and POFUT1 autoantibodies which were identified by SERPA were statistically different between ESCC or HGIN patients and HC in ELISA analysis with the area under the curve (AUC) values of 0.709 (95%CI: 0.654-0.764) and 0.741 (95%CI: 0.689-0.793), 0.717 (95%CI: 0.634-0.800) and 0.703 (95%CI: 0.627-0.779) for detection of ESCC and HGIN, respectively. Combining these two markers, the AUCs were 0.781 (95%CI: 0.733-0.829), 0.754 (95%CI: 0.694-0.814) and 0.756 (95%CI: 0.686-0.827) when distinguishing ESCC, early ESCC and HGIN from HC, respectively. Meanwhile, the expression of CETN2 and POFUT1 was found to be correlated with ESCC progression. Our data suggest that CETN2 and POFUT1 autoantibodies have potential diagnostic value for ESCC and HGIN, which may provide novel insights for early ESCC and precancerous lesions detection.

Identification and evaluation of novel serum autoantibody biomarkers for early diagnosis of gastric cancer and precancerous lesion.

Early diagnosis is crucial for optimal prognosis of gastric cancer (GC). Hereby, we aimed to identify novel serum autoantibody-based biomarkers for precancerous lesion (PL) and early GC. We performed serological proteome analysis (SERPA) combined with nanoliter-liquid chromatography combined with quadrupole time of flight tandem mass spectrometry (Nano-LC-Q-TOF-MS/MS) to screen for GC-associated autoantibodies. The identified autoantibodies were analyzed for potential detection value for PL and GC by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristic (ROC) curves analysis was conducted to evaluate the accuracy of the biomarkers. We identified seven candidates, such as mRNA export factor (RAE1), Nucleophosmin 1 (NPM1), phosphoglycerate kinase 1 (PGK1), and ADP-ribosylation factor 4 (ARF4). Antibodies against all seven proteins were present at higher levels in sera from 242 patients (51 PL, 78 early GC, 113 advanced GC) compared with sera from 122 healthy individuals. RAE1-specific autoantibody discriminated best between patients at different GC stages, with area under the curve (AUC) values of 0.710, 0.745, and 0.804 for PL, early GC, and advanced GC, respectively. Two predictive models composed of gender, RAE1, PGK1, NPM1, and ARF4 autoantibodies (Model 2 for PL) and of age, gender, RAE1, PGK1, and NPM1 autoantibodies (Model 3 for early GC) had improved diagnostic efficiencies, with AUCs of 0.803 and 0.857, sensitivities of 66.7% and 75.6%, and specificities of 78.7% and 87.7%, respectively. The identified serum tumor-associated autoantibodies (TAAbs) may have good potential for early detection of GC and PL.

Qualitative Analysis of Polyphenols in Glycerol Plant Extracts Using Untargeted Metabolomics.

Glycerol is a reliable solvent for extracting polyphenols from food and waste products. There has been an increase in the application of glycerol over benchmark alcoholic solvents such as ethanol and methanol for natural product generation because of its non-toxic nature and high extraction efficiency. However, plant extracts containing a high glycerol concentration are unsuitable for mass spectrometry-based investigation utilising electrospray ionization, inhibiting the ability to analyse compounds of interest. In this investigation, a solid phase extraction protocol is outlined for removing glycerol from plant extracts containing a high concentration of glycerol and their subsequent analysis of polyphenols using ultra-performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry. Using this method, glycerol-based extracts of Queen Garnet Plum (Prunus salicina) were investigated and compared to ethanolic extracts. Anthocyanins and flavonoids in high abundance were found in both glycerol and ethanol extracts. The polyphenol metabolome of Queen Garnet Plum was 53% polyphenol glycoside derivatives and 47% polyphenols in their aglycone forms. Furthermore, 56% of the flavonoid derivates were found to be flavonoid glycosides, and 44% were flavonoid aglycones. In addition, two flavonoid glycosides not previously found in Queen Garnet Plum were putatively identified: Quercetin-3-O-xyloside and Quercetin-3-O-rhamnoside.

Characterization and in vitro prebiotic activity analysis of succinoglycan produced by Rhizobium radiobacter

In this study, a novel succinoglycan (COGs-3) was produced and purified from Rhizobium radiobacter ATCC 13333, and its structural characteristics, physical properties, and in vitro prebiotic activity were investigated. The COGs-3 production was 2.761 g·L−1 after single-factor and response surface optimizations. The molecular weight and molecular formula of COGs-3 were 1584 Da and C59H92O49. The structure of COGs-3 consisted of β-Glcp-(1→3)-β-Glcp-(1→3)-β-Glcp-(1→6)-β-Glcp-(1→6)-β-Glcp-(1→4)-β-Glcp-(1→4)-β-Glcp-(1→3)-α-Galp modified with two succinate and one pyruvate groups based on ultraviolet–visible, Fourier transform infrared spectroscopy, high-performance gel filtration chromatography, elemental, monosaccharide composition, matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry, methylation, and nuclear magnetic resonance analyses, which was the type of succinoglycan. In addition, the thermal gravimetric and differential scanning calorimetry analyses showed that 40.23 % of COGs-3 maintained physical integrity when the temperature reached 600 ℃ and the melting point was 171.42 °C, which confirmed that COGs-3 had good thermal stability. The effects of COGs-3 on healthy human gut microbiota in vitro were also studied. COGs-3 was not digested by digestive juice and significantly increased the relative abundances of Bifidobacterium and Lactococcus and decreased the abundance of Megamonas and klebsiella. In addition, COGs-3 considerably promoted short-chain fatty acids biosynthesis. This work presents the first report on the in vitro prebiotic properties of succinoglycan, thereby providing new ideas for the application and development of succinoglycan.

Mechanism of famous classical formula Huaihua Powder in treatment of ulcerative colitis based on metabonomics

Ultra-high performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry(UHPLC-Q-TOF-MS) was employed in this study to observe the effect of Huaihua Powder on the serum metabolites of mice with ulcerative colitis and reveal the mechanism of Huaihua Powder in the treatment of ulcerative colitis. The mouse model of ulcerative colitis was established by dextran sodium sulfate salt(DSS). The therapeutic effect of Huaihua Powder on ulcerative colitis was preliminarily evaluated based on the disease activity index(DAI), colon appearance, colon tissue morphology, and the content of inflammatory cytokines such as tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1β(IL-1β). UHPLC-Q-TOF-MS was employed to profile the endogenous metabolites of serum samples in blank control group, model group, and low-, medium-, and high-dose Huaihua Powder groups. Multivariate analyses such as principal component analysis(PCA), partial least squares discriminant analysis(PLS-DA), and orthogonal partial least squares discriminant analysis(OPLS-DA) were performed for pattern recognition. Potential biomarkers were screened by Mass Profiler Professional(MPP) B.14.00 with the thresholds of fold change≥2 and P&lt;0.05. The metabolic pathways were enriched by MetaboAnalyst 5.0. The results showed that Huaihua Powder significantly improved the general state and colon tissue morphology of mice with ulcerative colitis, reduced DAI, and lowered the levels of TNF-α, IL-6, and IL-1β in serum. A total of 38 potential biomarkers were predicted to be related to the regulatory effect of Huaihua Powder, which were mainly involved in glycerophospholipid metabolism, glycine, serine, and threonine metabolism, mutual transformation of glucuronic acid, and glutathione metabolism. This study employed metabolomics to analyze the mechanism of Huaihua Powder in the treatment of ulcerative colitis, laying a foundation for the further research.

Ultrasound-assisted extraction of polyphenols from Chinese propolis

IntroductionPropolis is a beneficial bioactive food with rich polyphenols content. Nowadays, an increasing interest is attracted to the extraction of polyphenols from raw propolis. This study utilized the novel ultrasound-assisted approach for polyphenol extraction from Chinese propolis, aiming to improve its extraction yield and reveal the relevant mechanisms via extraction kinetic study as well as the compositional and structural analysis.MethodsThe optimum ultrasound-assisted extraction conditions were optimized according to the total phenolic content and total flavonoids content. Compositional and structural analysis were conducted using high performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry, high-performance liquid chromatography, Fourier-transform infrared spectroscopy (FT-IR) and scanning electron microscope (SEM).Results and discussionThe optimum ultrasound-assisted extraction conditions were as follows: ratio of liquid to solid, 60:1; ultrasound power, 135 W; ultrasound duration, 20 min. Under the optimum conditions, the antioxidant activities of the extract were increased by 95.55% and 64.46% by 2,2-diphenyl-1-picrylhydrazyl radical scavenging ability assay and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging ability assay, respectively, compared to those obtained by traditional maceration. The second-order kinetics model was employed to study the extraction process; it was found that ultrasound significantly accelerated the extraction of propolis and increased the maximum extraction volume of phenolic compounds. The qualitative and quantitative analysis of polyphenol compositions showed that ultrasound did not change the polyphenol types in the extract but it significantly improved the contents of various flavonoids and phenolic acids such as galangin, chrysin, pinocembrin, pinobanksin and isoferulic acid. Likewise, the FT-IR analysis indicated that the types of functional groups were similar in the two extracts. The SEM analysis revealed that the ultrasound-assisted extraction enhanced the contact areas between propolis and ethanol by breaking down the propolis particles and eroding the propolis surface.