Ultra-performance Liquid Chromatography Quadrupole Time-of-flight Mass Spectrometry Research Articles

In vitro characterization of S-23 metabolites produced by human liver microsomes, and subsequent application to urine after a controlled oral administration

The selective androgen receptor modulators are a recent class of anabolic agents, used to improve athletic performance. Among these molecules, there is (2 S)-N-(4-cyano-3-trifluoromethylphenyl)− 3-(3-fluoro-4-chlorophenoxyl)2-hydroxy-2-methyl-propanamide, commonly known as S-23. This molecule appeared very recently on the doping market. As a result, very few data are available in the literature, and nothing has been published about long-term effects of S-23. The authors focused on the detection of S-23 and its metabolites in human urine, following a single oral administration of approx. 8 mg to a volunteer, using standard ultra-performance liquid chromatography-triple quadrupole-mass spectrometry (UPLC-MS/MS), and ultra-performance liquid chromatography-quadrupole time of flight-mass spectrometry (UPLC-Q-TOF-MS). To the best of the authors knowledge, this seems to be the first study ever achieved on S-23. In vitro experiment was performed, using human liver microsomes, in order to investigate the potential CYP- and UGT-dependent S-23 metabolites. Four metabolites were produced, which were identified as hydroxy-S-23 (C18H12O4N2ClF4: m/z [M-H-] 431.0423); O-dephenylate-S-23 (C12H10O3N2F3: m/z [M-H-] 287.0647); S-23-glucuronide (C24H20O9N2ClF4: m/z [M-H-] 591.0794) and hydroxy-S-23-glucuronide (C24H20O10N2ClF4: m/z [M-H-] 607.0743). After consumption of S-23, the parent drug was detectable in hydrolyzed urine from 2 h post administration up to 28 days, with concentrations ranging between 0.5 and 93 ng/mL. In the urine, only one of the four metabolites identified in vitro was detected, hydroxy-S-23. This metabolite was detected up to 28 days. It does not seem to increase the window of detection of S-23 as the ratio between hydroxy-S-23 and the parent drug was always lower than 1. Another metabolite, dihydroxy-S-23, not identified in vitro, was identified in the urine of the volunteer. Hair sample, collected one month after the consumption of a single tablet, was negative for S-23 and hydroxy-S-23, with a LOQ at 0.1 pg/mg.

Rapid Identification of Characteristic Chemical Constituents of Panax ginseng, Panax quinquefolius, and Panax japonicus Using UPLC-Q-TOF/MS.

Saponins are the main active components in Panax ginseng C. A. Mey. (PG), Panax quinquefolius L. (PQ), and Panax japonicus C. A. Mey. (PJ), which belong to the genus Panax in the Araliaceae family. Because the chemical components in the three species are similar, they are often mixed and misused in functional foods and pharmaceuticals applications. Therefore, it is urgent to establish a method to quickly distinguish among PG, PQ, and PJ. Ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was combined with data postprocessing to identify the main characteristic fragments (CFs) and the related neutral losses (NLs) of protopanaxadiol (PPD), protopanaxatriol (PPT), oleanolic acid (OLE), and ocotillol- (OCO-) type saponins. By comparing the mass spectral data, it was possible to rapidly classify and identify saponins in PG, PQ, and PJ. A total of twenty-three chemical components were identified in the PG samples, twenty-three components were identified in the PQ samples, and twenty-seven components were identified in the PJ samples. Among them, OCO-type saponins were characteristic of PQ and PJ. Ginsenoside Rf, which was absent from PQ, allowed for differentiation between PQ and PJ. The CFs and NLs in the mass spectra of the characteristic components of PG, PQ, and PJ allowed for the rapid classification and identification of these species. Additionally, these results provide technical support for the quality evaluation of Chinese herbal medicine and for constructing a scientific regulatory system.

Colour, chemical compounds, and antioxidant capacity of Astragali Radix based on untargeted metabolomics and targeted quantification.

Astragali Radix has been used for over 2000 years in traditional Chinese medicine. Its secondary xylem "Jinjing" and secondary phloem "Yulan" are important for evaluating the quality of the Daodi medicinal material in China. However, its systematic characterisation has not been conducted. This study aims to investigate the colour, chemical compounds, and antioxidant capacity of the secondary xylem and phloem of Astragali Radix on the basis of untargeted metabolomics, broadening the application scope of Astragali Radix in food and pharmaceutical industries. The L*, a*, and b* of the secondary xylem and phloem were measured by colorimetry, and the chemical compounds were identified and quantified by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and high-performance liquid chromatography-diode array detector-evaporative light scattering detection. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays were conducted to evaluate their antioxidant capacity. Thirty-one compounds were identified by UPLC-Q-TOF-MS. The secondary xylem exhibited high parameter b*, flavonoid content, and antioxidant capacity, while the secondary phloem was rich in astragalosides. The colour parameters of well-defined type A significantly varied from those of the other types. Well-defined type A also exhibited the highest antioxidant activity and flavonoid content, followed by middle type A-like, middle type B-like, and yellow shading type B. The colour parameters, chemical compounds, and antioxidant capacity among the different transverse sections of secondary xylem and phloem varied. The yellow colour of secondary xylem was correlated to high flavonoid content and antioxidant activity, and well-defined type A of Astragali Radix had better quality than other types.

Rheum officinale Baill. Treats zebrafish embryo thrombosis by regulating NOS3 expression in the arginine biosynthesis pathway

BackgroundRheum officinale Baill. (ROB), as one of the traditional Chinese medicines for promoting blood circulation and removing blood stasis, has a wide range of pharmacological effects, such as cardiovascular protection, and has become a common drug in the clinical care of thrombosis. ObjectiveAlthough there are some pharmacological studies on ROB in the treatment of thrombotic diseases, the mechanism and material basis are still unclear. Based on the arginine biosynthesis signalling pathway, this research explored the target proteins and metabolites related to the intervention of ROB in thrombosis and expounded on the antithrombotic mechanism of ROB from the comprehensive perspectives of target prediction, intermediate metabolites and potential metabolic pathways. MethodsIn this research, ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) technology was used to qualitatively detect the chemical compounds of ROB, and the antithrombotic activity of ROB was evaluated by establishing a zebrafish model. The target function was predicted by network pharmacology, and differential metabolites were screened by metabolomics and multivariate statistical analysis methods. Correlation analysis of network pharmacology and metabolomics screening results was conducted to identify the potential pathway of ROB intervention in thrombosis, and the prediction results were further verified. ResultsROB significantly reduced the reactive oxygen species (ROS) staining intensity in zebrafish induced by phenylhydrazine (PHZ) and improved the inhibition rate of thrombosis. By constructing the “herb-disease-component-target” network, it was concluded that the active ingredients of ROB in treating thrombosis involved emodin, aloe-emodin and physcion, and the key targets included nitric oxide synthase 2 (NOS2) and nitric oxide synthase 3 (NOS3). A total of 341 differential metabolites in zebrafish with thrombosis were screened by partial least squares discriminant analysis (PLS-DA). The results of reverse transcription-polymerase chain reaction (RT–PCR) experiments and targeted metabolomics verification showed that ROB was mainly involved in improving thrombosis by upregulating the expression of NOS3 mRNA and regulating the levels of arginine, glutamate and glutamine in the arginine biosynthesis pathway. ConclusionsROB improved thrombosis by regulating the expression of NOS3 mRNA and the contents of arginine, glutamate and glutamine in the arginine biosynthesis signalling pathway.

Weed Suppression and Molecular Mechanisms of Isochlorogenic Acid A Isolated from Artemisia argyi Extract via an Activity-Guided Method.

Allelopathy is considered an environmentally friendly and resource-conserving approach to weed control because allelochemicals degrade easily and cause less pollution than traditional chemical herbicides. In this study, the allelopathic active constituents of Artemisia argyi were elucidated by activity-guided isolation and ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). First, a crude extract prepared in water was fractionated using macroporous resin D101 to obtain three fractions (Fr.A-C). Combined with the allelopathic activity assay on Setaria viridis and Portulaca oleracea, Fr.C was determined to be the most active fraction. We identified 14 compounds in the active fraction (Fr.C) using UPLC-QTOF-MS, including 13 phenolic compounds. Accordingly, phenolic components have been suggested as the main allelochemicals in A. argyi. Thereafter, Fr.C was further isolated by octadecylsilyl (ODS) chromatography to obtain eight subfractions (Fr.C-1-Fr.C-8). Finally, isochlorogenic acid A (ICGAA) was purified from Fr.C-3 by semipreparative liquid chromatography, which was detected in the growth environment of A. argyi. Furthermore, we evaluated the allelopathic effects of ICGAA on six weeds from different families and genera for the first time. The results showed that ICGAA is a novel allelochemical with broad herbicidal activity. In addition, we analyzed the inhibitory effect and molecular mechanism of ICGAA on the growth of S. viridis seedlings. Optical microscopy and transmission electron microscopy (TEM) revealed the degradation of membrane structures and organelles after ICGAA treatment. Transcriptome and real-time polymerase chain reaction (RT-qPCR) analysis showed that ICGAA inhibited the growth of weeds mainly by inhibiting the diterpenoid biosynthesis pathway (especially gibberellins, GAs). The decrease of gibberellin (GA) contents after ICGAA treatment also confirmed these results. In brief, this study provides new material sources and theoretical support for developing biological herbicides for agroecosystems.

Comparative Elucidation of Age, Diameter, and "Pockmarks" in Roots of Paeonia lactiflora Pall. (Shaoyao) by Qualitative and Quantitative Methods.

Paeonia lactiflora Pall. is a world-famous ornamental plant, whose roots have been used as an important traditional Chinese medicine, Shaoyao, to treat diseases for more than 1,000 years. Because of the excellent curative effect of Shaoyao, its quality has attracted wide attention, however, there is a lack of comprehensive research on the different influencing factors of quality of Shaoyao. In this study, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) and high-performance liquid chromatography with diode-array detection (HPLC-DAD) were utilized to systematically analyze the Shaoyao of different ages, diameters and roots with “pockmarks.” 60 metabolites were detected and identified from Shaoyao using the UPLC-Q/TOF-MS, of which 20 potential quality markers of dissected roots with and without “pockmarks” were selected for the first time using the orthogonal partial least squares discriminant analysis (OPLS-DA) and the variable importance for projection (VIP) plot. Then, a selective and accurate HPLC-DAD quantitative assay has been developed for the simultaneous determination of 11 bioactive components in Shaoyao. The results showed that the total content of five monoterpene glycosides including oxypaeoniflorin, albiflorin, paeoniflorin, lactiflorin, and benzoylpaeoniflorin and six phenols including gallic acid, catechin, methyl gallate, ethyl gallate, apiopaeonoside and benzoic acid in the 3-year-old Shaoyao was higher than that of 4-year-old and 5-year-old Shaoyao. In Shaoyao of the same age, the total content of five monoterpene glycosides and six phenols decreased with an increase in diameter. In addition, regardless of whether it is a whole or a divided root, the contents of five monoterpene glycosides and six phenols in Shaoyao with “pockmarks” were higher than those of Shaoyao without “pockmarks.” In summary, this work has explored several factors that might affect the quality of Shaoyao, and provide a guide for more comprehensive quality evaluation in its further production, processing, and rational utilization.

Identification of Common Liver Metabolites of the Natural Bioactive Compound Erinacine A, Purified from Hericium erinaceus Mycelium

Metabolite identification, in the early stage, for compound discovery is necessary to assess the knowledge for the pharmaceutical improvement of drug safety and efficacy. Even if the drug has been released into the market, identification and continuous evaluation of the metabolites are required to avoid the risk of post-marketing withdrawal. Hericium erinaceus (HE), a medicinal mushroom, has broadly documented nutraceutical benefits, including anti-oxidant, anti-tumor, anti-aging, hypolipidemic, and gastric mucosal protection effects. Recently, erinacine A has been reported as the main natural bioactive compound in the mycelium of HE for functional food development. In neurological studies, the consumption of enrinacine A enriched HE mycelium demonstrates its significant nutraceutical effects in Alzheimer’s disease, Parkinson’s disease, and ischemic stroke. For the first time, we explored the metabolic process of erinacine A molecule and identified its metabolites from the rat and human liver S9 fraction. Using a liquid chromatography/triple quadrupole mass spectrometer for quantitative analysis, we observed that 75.44% of erinacine A was metabolized within 60 min in rat, and 32.34% of erinacine A was metabolized within 120 min in human S9. Using an ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) to identify the metabolites of erinacine A, five common metabolites were identified, and their possible structures were evaluated. Understanding the metabolic process of erinacine A and establishing its metabolite profile database will help promote the nutraceutical application and discovery of related biomarkers in the future.

Chemical composition and antibacterial activity of 12 medicinal plant ethyl acetate extracts using LC-MS feature-based molecular networking.

Widespread use of antibiotics has led to an increase in bacterial multiple drug resistance, thereby searching for natural antimicrobial agents from plants becomes an effective and alternative approach. In the present study, we selected six foodborne bacteria to evaluate the antibacterial activities of 12 medicinal plants ethyl acetate (EA) extracts. This study aims to search for natural antibiotic substitutes from plant extracts. The antibacterial components were further discussed through chemometric and mass spectroscopic analyses. Agar well diffusion and the microdilution methods were used to test the antibacterial activity. Total phenolic content (TPC) and total flavonoid content (TFC) were used to judge the active phytochemicals. To further characterise the potential antibacterial components, an ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS) coupled with Pearson correlation and feature-based molecular network (FBMN) were proposed. Most of the plant extracts possessed antibacterial activity against Bacillus subtilis and Salmonella typhi. Toona sinensis shoots and Firmiana simplex barks showed high inhibitory activities against Staphylococcus aureus, Shigella dysenteriae, and Escherichia coli strains with minimum inhibitory concentrations (MICs) of 1.56, 0.78, and 0.39 mg/mL, respectively. Salmonella typhi was highly sensitive to Firmiana simplex barks with an inhibitory diameter up to 21.67 ± 0.95 mm, and MIC at 0.78 mg/mL. Moreover, Toona sinensis shoots and Firmiana simplex barks had the highest TPCs. Our results indicated that Toona sinensis shoots, Koelreuteria paniculate seeds, and Firmiana simplex barks could be supplied as potential sources of antimicrobial agents. Furthermore, 36 potential bioactive compounds were identified mainly as polyphenols, glycosides, and terpenoids.

Comparison of the chemical composition and antioxidant, anti-inflammatory, α-amylase and α-glycosidase inhibitory activities of the supernatant and cream from black tea infusion.

Tea cream is a kind of turbid substance commonly existing in tea infusion and tea beverage upon cooling. Herein, a comparative study was conducted on the supernatant and cream from black tea infusion in terms of antioxidant, anti-inflammatory and enzyme inhibitory activities, and chemical composition. Ultraviolet-visible (UV-vis) spectrometry and high-performance liquid chromatography (HPLC) analysis showed that the contents of protein, polyphenols, theaflavins, thearubigins, theabrownins, and caffeine in cream were significantly higher than those in the supernatant. The contents of Al, Ca, Cu, and Fe elements in cream were higher than those in the supernatant. However, higher levels of monosaccharides and free amino acids were detected in the supernatant compared with cream. The ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) based metabolomics analysis revealed that the main marker compounds between the supernatant and the cream were organic acids, phenolic acids, and flavan-3-ols and their oxidation products, flavonol glycosides and amino acids. The cream showed better antioxidant and anti-inflammatory, as well as α-amylase and α-glycosidase inhibitory activities than the supernatant, because it contained higher contents of polyphenols than the supernatant. The present study expanded the new vision towards the cream of black tea infusion.

Screening and identification of differential metabolites in serum and urine of bamaxiang pigs bitten by <i>trimeresurus stejnegeri</i> based on UPLC-Q-TOF/MS metabolomics technology

Trimeresurus stejnegeri is one of the top ten venomous snakes in China, and its bite causes acute and severe diseases. Elucidating the metabolic changes of the body caused by Trimeresurus stejnegeri bite will be beneficial to the diagnosis and treatment of snakebite. Thus, an animal pig model of Trimeresurus stejnegeri bite was established, and then the metabolites of serum and urine were subsequently screened and identified in both ESI+ and ESI- modes identified by ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS) methods. There are 9 differential metabolites in serum, including Oleic acid, Lithocholic acid, Deoxycholic acid, Hypoxanthine, etc. There are 11 differential metabolites in urine, including Dopamine, Thiocysteine, Arginine, Indoleacetaldehyde, etc. Serum enrichment pathway analysis showed that 5 metabolic pathways, including Tryptophanuria, Liver disease due to cystic fibrosis, Hartnup disease, Hyperbaric oxygen exposure and Biliary cirrhosis, the core metabolites in these pathways, including deoxycholic acid, lithocholic acid, tryptophan and hypoxanthine, changed significantly. Urine enrichment pathway analysis showed that 4 metabolic pathways, including Aromatic L-Amino Acid Decarboxylase, Vitiligo, Blue Diaper Syndrome and Hyperargininemia, the core metabolites in these pathways including dopamine, 5-hydroxyindole acetic acid and arginine. Taken together, the current study has successfully established an animal model of Trimeresurus stejnegeri bite, and identified the metabolic markers and metabolic pathways of Trimeresurus stejnegeri bite. These metabolites and pathways may have potential application value and provide a therapeutic basis for the treatment of Trimeresurus stejnegeri bite.

UPLC‐Q‐TOF/MS‐based metabonomics study on Apostichopus Japonicus in various aquaculture models

Apostichopus japonicus is an important economic species with a high nutritional value. Aquaculture models have diversified with the rapid development of the sea cucumber industry, resulting in a distinct quality of A. japonicus. In this study, ultra-performance liquid chromatography–quadrupole–time-of-flight mass spectrometry (UPLC-Q-TOF/MS) technology was used to analyse body wall metabolites of cage-cultured, pond-cultured and bottom-sowing A. japonicus. The results were combined with multivariate analysis (PLS-DA) and KEGG to identify the differential metabolites under various aquaculture models. The results revealed that cage-cultured A. japonicus showed a high level of lipid metabolism, including Docosapentaenoic Acid (DPA), Adrenic Acid, Docosahexaenoic Acid (DHA), PC (2:0/19:2). Pond-cultured A. japonicus showed a high level of metabolism of LysoPC 16:1, LysoPC 18:3, LysoPC 20:5, Isoleucine, 3-Dehydroxycarnitine. Bottom-sowed A. japonicus showed a high level of metabolism of Leucenol, Uridine, LysoPC 22:1 and Inosine. KEGG pathway analysis showed that the differential metabolic pathways between cage- and pond-cultured A. japonicus were in lipid metabolism, amino acid metabolism and carbohydrate metabolism. Differential metabolic pathways between cage-cultured and bottom-sowed A. japonicus were in lipid metabolism and nucleotide metabolism. The differential metabolic pathways between pond-cultured and bottom-sowed A. japonicus were in lipid metabolism and nucleotide metabolism. The results of this study provide new knowledge for further understanding metabolic differences of A. japonicus in various aquaculture models. Moreover, the data may provide meaningful guidelines for A. japonicus farming and consumption.

Screening and identification of acetylcholinesterase inhibitors from Terminalia chebula fruits by immobilized enzyme on cellulose filter paper coupled with ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry and molecular docking

With the increasing demand of new drugs for the treatment of Alzheimer's disease (AD), screening acetylcholinesterase (AChE) inhibitors from traditional Chinese medicines (TCMs) has been proved to be an effective strategy for drug discovery. In present study, a novel strategy was developed to fish out AChE inhibitors from Terminalia chebula fruits based on immobilized AChE coupled with ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) and molecular docking. For AChE immobilization, cellulose filter paper (CFP) as the carrier was modified with chitosan to be introduced to amino groups, and then AChE was modified on the amino-modified CFP through a Schiff base reaction with glutaraldehyde as a cross-linking agent. The CPF-immobilized AChE possessed advantages of a wider range for pH and temperature endurance, better storage stability, excellent reproducibility and reusability. The CPF-immobilized AChE was incubated with the extract of T. chebula fruits, and then the active components would form complexes with immobilized AChE. The complexes were further conveniently separated with inactive components by virtue of the instantaneous separation characteristic of CFP. Eventually, 25 (1–11, 13–26) potential AChE inhibitors were fished out and their structures were further identified by UPLC-QTOF-MS. Moreover, molecular docking was performed to discriminate non-specific compounds to AChE and explore binding mechanisms between potential inhibitors and AChE, and 25 compounds could be well embedded into active sites of AChE with affinities ranging from -9.9 to -6.4 kcal/mol. Inhibitory activities of screened active components on AChE were evaluated in vitro, and punicalagin, 1,3,6-tri-O-galloyl-β-D-glucose (1,3,6-TGG), chebulinic acid and geraniin exhibited excellent AChE-inhibitory properties with IC50 values of 0.43 ± 0.03, 0.46 ± 0.02, 0.50 ± 0.03 and 0.51 ± 0.03 mM, respectively. The results indicated that the developed method was simple and efficient, and could be utilized to screen and identify potential AChE inhibitors from TCMs.

Study on Chinese patent medicine based on major component analysis and quality control evaluation: A case study of Jizhi Syrup

Jizhi Syrup (JZS) is a popular Chinese patent medicine (CPM) for the treatment of respiratory diseases in clinical practice, especially acute or chronic bronchitis. JZS is a complex formula composed of 8 kinds of herbs and lack of comprehensive researches on chemical components. To further define its components, ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS) and headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) were utilized to identify and classify the chemical components of JZS. A total of 178 chemical compounds encompassing the 8 herbs of JZS were identified and the chemical components were comprehensively explicit. It made up for the gap that volatile components were not studied in the previous study. Based on this, a new method for the quality control of JZS based on its characteristic components was established by fingerprints, multi-component quantitative analysis and quantity transfer of JZS. A dual-wavelength high-performance liquid chromatography (HPLC) fingerprints were established at 210 nm and 260 nm. Four volatile components (linalool, bornyl acetate, 2-undecanone and α-terpineol) and eight nonvolatile components (ephedrine hydrochloride, protocatechuic acid, 5-caffeoylquinic acid, 4-hydroxybenzoic acid, naringin, neohesperidin, glycyrrhizic acid and praeruptorin A) were quantitated by HS-SPME-GC-MS and HPLC-diode array detection (DAD). Meanwhile, six exclusive nonvolatile components were studied for the quantity transfer of Herbs-Intermediate-CPM and all the transfer rates were between 55.23% and 89.20%. This study is the first comprehensive study of the major components in JZS, and its results can be useful to standardize the quality control and provide a valuable reference for other CPMs.

A novel strategy for the discovery and validation of allergic component and its action mechanism in Red Ginseng

Red ginseng (a processed product of ginseng) is a famous herbal medicine or healthcare product widely used in Asia. However, there are many reports about anaphylactoid reaction of red ginseng. This study aims to establish a novel strategy for discovering and validating allergic component and its action mechanism in red ginseng. Firstly, RBL-2H3 cell degranulation experiment was performed by real-time cell analysis (RTCA) system and β-hexosaminase release test to illustrate the anaphylactoid reaction induced by red ginseng. Then, combined with ultra-performance liquid chromatography quadrupole/time of flight mass spectrometry (UPLC-Q/TOF-MS) analysis, the main components of red ginseng for inducing degranulation of RBL-2H3 cells was explored. Furthermore, the compounds with the parent nucleus structure of allergic component were studied to figure out whether they could induce RBL-2H3 cell degranulation, and the relationship between the structures of compounds and degranulation was further investigated. Finally, the underlying mechanism of allergic components was explored based on network toxicology and molecular docking method, which was further verified by western blotting analysis. Our findings demonstrated that red ginseng extract could induce degranulation of RBL-2H3 cells, and ginsenoside Ro (an oleanane-type triterpene saponin), was confirmed to be the main allergic component. Meanwhile, both ginsenoside Ro (Ro) and other oleanane-type triterpene saponins could induce degranulation of RBL-2H3 cells, and the compound with disaccharide chain on C3 position had the strongest activity of degranulation. And their mechanism of inducing degranulation of RBL-2H3 cells involved in PI3K-Akt pathway. The results would provide a reference for the quality control and evaluation of safety of herb medicines or healthcare products containing oleanane-type triterpenoid saponins. Our method offers a novel strategy for screening allergic components in red ginseng and promoting further study of safety evaluation of red ginseng.

Protective effects of Fufang Ejiao Jiang against aplastic anemia assessed by network pharmacology and metabolomics strategy

ObjectiveTo elucidate the mechanisms underlying the therapeutic effects of Fufang Ejiao Jiang (复方阿胶浆, FFEJJ) on aplastic anemia (AA) using integrated network pharmacology and serum metabolomics. MethodsTraditional Chinese Medicine Systems Pharmacology (TCMSP), Pubmed, integrative pharmacology-based research platform of traditional Chinese medicine (TCMIP), and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN-TCM) were used to identify the constituents and putative targets of FFEJJ. GeneCards and DisGeNET databases were used to identify AA-associated targets. We constructed a herb-component-target network and analyzed the protein-protein interaction (PPI) network. Potential mechanisms were determined using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. In addition, an AA model was established using acetylphenylhydrazine (APH) and cetylphenylhydrazine (CTX). Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS)-based serum metabolomics was applied to screen potential metabolites and the related pathways associated with AA and the potential anti-anemic effects of FFEJJ. ResultsA total of 30 active components of FFEJJ and 24 targets were related to AA. PPI network analysis showed that VEGFA, AKT1, IL-6, CASP3, and ICAM1 were key nodes overlapping with proteins known to be related to AA. KEGG pathway enrichment analysis revealed that the presumed targets of FFEJJ were mainly associated with pathways linked to the promotion of hematopoiesis and improvement of the hematopoietic microenvironment. A total of 423 metabolite biomarkers were identified between the control and AA models, which are involved in the development of AA. In contrast, FFEJJ reversed the 79 differential metabolites altered by AA. Pathway analysis suggested that the synergistic effects of FFEJJ were mainly enriched in 24 metabolic pathways. Among them, sphingolipid metabolism, glycerophospholipid metabolism, and arachidonic acid metabolism were related to promoting hematopoiesis and improving the hematopoietic microenvironment, which partially conforms with network pharmacology. The interaction network formed by three key differential metabolites, including hydroxy-eicosatetraenoic acid (HETE), sphingosine 1-phosphate (S1P), and lysophosphati-dylcholine (lysoPC), and three predicted network targets (VEGFA, CASP3, and ICAM1) may be the potential mechanism underlying the anti-AA action of the multi-component of FFEJJ. ConclusionFFEJJ could be an alternative treatment option for AA. It acts by promoting hematopoiesis and improving the hematopoietic microenvironment. Network pharmacology-integrated metabolomics makes it possible to analyze TCMs from a systems perspective and at the molecular level.

2D HILIC‐ELSD/UPLC‐Q‐TOF‐MS Method for Acquiring Phospholipid Profiles and the Application in Caenorhabditis elegans

AbstractPhospholipids are the main constituent of cellular membranes and have recently been identified to have diagnostic value as biomarkers for many diseases. Accordingly, much emphasis is now laid on developing optimal analytical techniques for the phospholipid profiles of various biological samples. In the present study, different classes of phospholipids are first separated by optimized hydrophilic interaction chromatography with evaporative light scattering detector (HILIC‐ELSD). The phospholipids in each class are then identified by ultraperformance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS). Validation results confirm that this approach meets the requirements of quantitative analysis. Finally, the approach is adopted to analyze the phospholipid profiles in Caenorhabditis elegans. A total of 111 phospholipid species is identified according to the mass fragments. Major fatty acyl chains in phospholipids are found to be formed by oleic acid (C18:1), arachidonic acid (C20:4), and eicosapentaenoic acid (C20:5). Overall, this study improves current knowledge on analytical techniques of the phospholipid composition in C. elegans and provides a basis for future lipidomics research.Practical applications: Phospholipids reportedly play a crucial role in the development of many diseases. Until now, only a small portion of phospholipids in Caenorhabditis elegans has been reported by using one‐dimensional analysis strategy. The offline 2D liquid chromatography method developed in this study identifies 111 phospholipid species in Caenorhabditis elegans. The obtained phospholipid profiles complement the lipid database of Caenorhabditis elegans. The study also provides the basis for the future development of a 2D online approach.

Application of UPLC-QTOF-MS non-targeted metabonomics in mechanism study of property differences of ginseng and American ginseng

This study adopted ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS)-based untargeted metabolomic approaches for exploring the changes in endogenous metabolites of rat serum related to property differences between ginseng and American ginseng. Then the action mechanisms of them with warm and cool properties and the effects of processing on their property changes were investigated. Based on principal component analysis(PCA), the differences in metabolite profiles between ginseng, red ginseng, American ginseng, and red American ginseng were compared. After that, 16 potential differential endogenous biomarkers were identified by orthogonal partial least squares discriminant analysis(OPLS-DA) and online database searching. And the related metabolic pathways were systematically analyzed. By comparing content variations of these 16 potential differential endogenous biomarkers, we have found that 10 potential differential biomarkers were responsible for the warm property of ginseng and red ginseng, and 9 were related to the cool property of American ginseng and red American ginseng. As demonstrated by in-depth analysis of related metabolic pathways of differential biomarkers, ginseng and American ginseng mainly played a role in regulating the energy metabolism of amino acid, glycolysis, and fatty acids, during which they exhibited differences in property. The comparison of content variations of these differential endogenous between groups revealed that the energy metabolism of red ginseng group was stronger than that of ginseng group, consistent with the traditional processing theory that the warming and tonifying effects of ginseng could be enhanced after processing. The property of red American ginseng was similar to that of American ginseng, both cool in property, but American ginseng was cooler than red American ginseng. It can be seen that non-targeted metabolomic approaches can be utilized to study mechanisms underlying property differences of Chinese medicines and the effects of processing on their property changes.

Eggshell Valorization: Membrane Removal, Calcium Oxide Synthesis, and Biochemical Compound Recovery towards Cleaner Productions

As climate change continues to rank high among issues of global concern, industries such as agriculture and construction continue to unearth possible ways to curb carbon dioxide generation and encourage the use or reuse of a variety of by-products and waste materials, fostering the implementation of cleaner technologies. Eggshells form a notable component of this waste, making up more than 7.6 million metric tonnes annually. Research works involving the calcination of eggshells have often been done by burning both shell and its constituent proteins and membrane to produce calcium oxide, CaO. This novel research investigated a cleaner means of CaO synthesis by recovering the shell membrane and some valuable chemical compounds from eggshells before calcination. Atomic absorption (AA), thermogravimetric analysis (TGA), scanning electron microscope (SEM), X-ray fluorescence analysis (XRF), X-ray diffraction (XRD), Ultra-performance-liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS), and RGB color analysis were all employed. Acetic and Nitric acid was used to weaken the shell-membrane bond, thereby aiding membrane separation. Shell membrane was easily separated after 17 minutes of soaking time. Calcium oxide, CaO was synthesized from separated shells after calcination for 3 hours at 900 ℃. 99% CaO with an RGB value of 253 was produced. Collagen, as well as other chemical compounds, were recovered. Eggshell was successfully valorized for CaO production. The shell membrane, collagen, and other recovered compounds, which would have been burnt off and left as an impurity in the CaO, can now the put to more profitable use.

Metabolomics study of Angelica sinensis (Oliv.) Diels on the abnormal uterine bleeding rats by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry analysis.

The objective of this study was to explore the effects and underlying intervention mechanisms of Angelica water extract (AWE) on abnormal uterine bleeding (AUB) based on serum metabolomics. Firstly, the concentration of main active substances in AWE was determined and the chemical components were identified by UPLC‐Q‐Exactive Orbitrap‐MS/MS. A drug‐induced abortion model was established by mifepristone and misoprostol. After administration AWE (2.16 g/kg) for 7 days, the coagulation function, serum hormone levels, H&E staining, and immunohistochemistry observation of uterus were detected. In addition, serum metabolites profiles were performed on ultra‐performance liquid chromatography–quadrupole–time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS). The contents of ferulic acid, senkyunolide A, and ligustilide in AWE were 0.7276, 0.0868, and 1.9908 mg/g, respectively. Twenty‐six compounds were identified in AWE. It was found that AWE was effective in regulation of coagulation function and promoting endometrial recovery. Meanwhile, the levels of E2, Pg, and HCG and the expression of ERα, Erβ, and PR were down‐regulated in AUB model and up‐regulated by the treatment of AWE. Twenty‐one potential biomarkers were eventually identified by multivariate statistical analysis. Study indicated that glycerophospholipid, sphingolipid, amino acids, retinol metabolism and primary bile acid biosynthesis were the main related metabolic pathways involved for the treatment of AUB by AWE. The results showed that AWE has potential therapeutic effect on AUB by altering the metabolic aberrations.

Untargeted Metabolomics and Targeted Quantitative Analysis of Temporal and Spatial Variations in Specialized Metabolites Accumulation in Poria cocos (Schw.) Wolf (Fushen).

Poria cocos (Schw.) Wolf is a saprophytic fungus that grows around the roots of old, dead pine trees. Fushen, derived from the sclerotium of P. cocos but also containing a young host pine root, has been widely used as a medicine and food in China, Japan, Korea, Southeast Asian countries, and some European countries. However, the compound variations at the different growth periods and in the different parts of Fushen have not previously been investigated. In this study, an untargeted metabolomics approach based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) and targeted quantitative analysis was utilized to characterize the temporal and spatial variations in the accumulation of specialized metabolites in Fushen. There were 119 specialized metabolites tentatively identified using the UPLC-Q/TOF-MS. The nine growth periods of Fushen were divided into four groups using partial least squares discrimination analysis (PLS-DA). Four different parts of the Fushen [fulingpi (FP), the outside of baifuling (BO), the inside of baifuling (BI), and fushenmu (FM)] were clearly discriminated using a PLS-DA and orthogonal partial least squares discrimination analysis (OPLS-DA). Markers for the different growth periods and parts of Fushen were also screened. In addition, the quantitative method was successfully applied to simultaneously determine 13 major triterpenoid acids in the nine growth periods and four parts. The quantitative results indicated that the samples in January, March, and April, i.e., the late growth period, had the highest content levels for the 13 triterpenoid acids. The pachymic acid, dehydropachymic acid, and dehydrotumulosic acid contents in the FM were higher than those in other three parts in March, whereas the poricoic acid B, poricoic acid A, polyporenic acid C, dehydrotratrametenolic acid, dehydroeburicoic acid, and eburicoic acid in FP were higher beginning in October. These findings reveal characteristics in temporal and spatial distribution of specialized metabolites in Fushen and provide guidance for the identification of harvesting times and for further quality evaluations.