High Performance Liquid Chromatography Quadrupole Research Articles

The screening method for 39 phytotoxins and mycotoxins in blood and urine with liquid chromatography-high resolution mass spectrometry

BackgroundPoisonings caused by plant toxins and mycotoxins occur frequently, which do great harm to human health and social public health safety. When a poisoning incident occurs, biological samples are commonly be used to conduct the detection of toxic substances and their metabolites for targeted clinical treatment and incident analysis. ObjectiveTo establish an efficient and accurate analysis method of 39 phytotoxins and mycotoxins in blood and urine by high performance liquid chromatography quadrupole tandem orbitrap mass spectrometry (HPLC-Orbitrap MS). MethodAfter 3 mL of methanol being added to 1 mL blood and urine respectively for extraction and protein precipitation, the supernatant was injected into HPLC-Orbitrap MS for analysis. The phytotoxins and mycotoxins were separated by Hypersil GOLD PFP column with gradient elution using methanol-5 mmol/L ammonium acetate as mobile phase. The data were collected in ESI positive ion mode using Full MS/dd-MS2 for mass spectrometry detection. ResultThe mass database of 39 phytotoxins and mycotoxins was developed, and accurate qualitative analysis can be obtained by matching with the database using the proposed identification criteria. Limit of detections (LODs) were 1.34 × 10−4 ∼ 1.92 ng/mL and 1.92 × 10−4 ∼ 9.80 ng/mL for blood and urine sample, respectively. Limits of quantification (LOQ) of toxins in blood and urine ranged from 4.47 × 10−4 ∼ 6.32 ng/mL and 6.39 × 10−4 ∼ 32.67 ng/mL, respectively. Intra-day relative standard deviations (RSDs) were 0.79 % ∼ 10.90 %, and inter-day RSDs were 1.08 % ∼ 18.93 %. The recoveries can reach 90 % ∼ 110 % with matrix matching calibration curves. ConclusionThe established method is simple and rapid to operate, which can complete the sample analysis within 30 min, providing technical support for clinical poisoning treatment and public health poisoning analysis.

High-Performance Liquid Chromatography–Quadrupole Time-of-Flight Tandem Mass Spectrometry-Based Profiling Reveals Anthocyanin Profile Alterations in Berries of Hybrid Muscadine Variety FLH 13-11 in Two Continuous Cropping Seasons

FLH 13-11 is an F1 interspecific hybrid muscadine grape genotype that was developed to produce new anthocyanins for pigment color stability. This hybrid resulted from a cross between ‘Marsh’ (Vitis munsoniana) and ‘Magoon’ (V. rotundifolia) and has been cultivated for the wine and juice industry. This report characterizes anthocyanins produced in fully ripe berries and reveals a significant difference in total anthocyanin contents from two continuous cropping seasons. High-performance liquid chromatography with a diode array detector (HPLC-DAD) and HPLC–quadrupole time-of-flight tandem mass spectrometry (HPLC-qTOF-MS/MS) were used to profile anthocyanins in berries. The resulting data showed that fourteen anthocyanins were detected, six from 2011 and nine from 2012, with only one produced in both seasons. However, the anthocyanidin profiles of the berries were the same. Five anthocyanins were annotated as diglucosides of anthocyanidins based on MS/MS features, including delphinidin 3,5-diglucoside produced in both seasons, cyanidin 3,5-diglucoside mainly formed in 2011, petunidin 3,5-diglucoside, malvidin 3,5-diglucoside, and peonidin 3,5-glucoside only detected in 2012. Also, three anthocyanidin-diglucoside-like anthocyanins and three monoglucosides, including peonidin 3-glucoside, delphinidin 3-glucoside like, and pelargonidin 3-glucoside-like anthocyanins, were detected in 2011 and 2012, respectively. These results indicate that FLH 13-11 can produce both anthocyanidin-diglucosides and -monoglucosides, and their biosynthesis is closely dependent on cropping years.

Profiling of 3-Formyl rifamycin (3-FR) from sintered fixed dose combinations (SDCs) of rifampicin and isoniazid by using RP-HPLC, LC-ESI-QTOF and UPLC/MS/MS

The rapid and severe decomposition of rifampicin in the presence of isoniazid (INH) in a gastric acidic pH environment is a well-reported phenomenon. It is important to note that the significant decomposition problem of RIF has an impact on its bioavailability, which becomes a major issue in fixed dose combinations (FDCs). In this degradation process, RIF will undergo acid hydrolysis and form 3-FR, an acid degradation product of RIF. Further, it will react with INH and form an insoluble iso-nicotinoyl hydrazone (HYD). The formed HYD is probably not stable in the acidic condition, so it will further convert into INH and 3-FR. Consequently, the generation of 3-FR in the acidic stomach environment can be a critical factor contributing to a substantial decline in RIF bioavailability and warrants careful consideration. Hence, it is imperative to explore the presence of 3-FR not only in in vitro dissolution samples but also in in vivo plasma samples. Therefore, the current study focused on the chemical analysis of 3-FR using additively manufactured sintered drug combinations (SDCs) to evaluate its formation, release and qualitative absorption in both in vitro and in vivo settings. High-performance liquid chromatography (HPLC) and liquid chromatography-electrospray ionization quadrupole time-of-flight high-resolution mass spectrometry (LC-ESI-QTOF-MS/MS) were employed for analytical investigations. The developed LC/MS-MS method was employed to investigate the presence of 3-FR in the rabbit's plasma following a single oral administration of SDCs. The study revealed the significant presence of 3-FR in both the in vitro and in vivo plasma samples obtained from the SDCs of RIF and INH. This finding indicates the potential of altering the strategy through additive manufacturing of pH-dependent SDCs to address the critical issue of RIF.

Nitrogen-doped carbon enhances Fe0 activation for efficient tetrabromobisphenol a decontamination and its removal mechanism

Biomass derived N-doped carbon-encapsulated nano zero-valent iron (NC@Fe0) was successfully used to activate hydrogen peroxide (H2O2) for efficient oxidation of tetrabromobisphenol A (TBBPA). The as-prepared NC@Fe0 exhibited high H2O2 activation performance, with a TBBPA removal efficiency of 95.9 % and an oxidation rate constant of 0.031 min−1. The quenching experiments and electrochemical tests showed that the overall contributions of nonradical pathways were over 83.0 %, via synergistic production of singlet oxygen (1O2) and an electron transfer process (ETP). Specifically, the N group on NC@Fe0 greatly enhanced 1O2 production, while formation of surface complexes (OVs-H2O2) was responsible for the ETP mechanism. High Performance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometer (HPLC-Q-TOF MS/MS) and Toxicity Estimation Software Tool (T.E.S.T) clearly demonstrated that products were mainly generated via debromination and β-scission, where the products were less toxic than the parent. During practical wastewater, in both lake water and domestic wastewaters, NC@Fe0/H2O2 removed over 91.0 % of TBBPA. This study highlighted the considerable contribution of nonradicals in contaminant removal and enriched our understandings of a green activator for future environmental remediations.

Investigating the mechanism and efficacy material basis of Xiehuo Xiaoying decoction for treating Graves' disease via thyroid cell apoptosis based on proteomics and molecular docking techniques

Ethnopharmacological relevanceFor numerous years, the Xiehuo Xiaoying decoction (XHXY), a traditional Chinese medicine formula, has demonstrated substantial promise in treating Graves' disease (GD) in clinical settings, showcasing significant potential. However, the therapeutic mechanism and efficacy material basis of XHXY remains obscure. Aim of the studyThis work aims to investigate the underlying mechanisms and to study the efficacy material basis of XHXY in anti-GD effect using a combination of TMT quantitative proteomics and molecular docking method. Materials and methodsGD model was initiated by administering Ad-TSH289. Subsequently, the mice underwent a four-week regimen that included oral gavage of XHXY at doses of 17 g/kg·d and 34 g/kg·d, along with intraperitoneal injections of Gentiopicroside (GPS). Utilizing the principles of pharmacological chemistry in traditional Chinese medicine, we employed high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-QTOF/MS) to discern prescribed prototype composition of XHXY in serum samples from mouse. TMT proteomics research provided evidence of XHXY's putative targets and important pathways in vivo. The binding activity of probable action targets and prototype composition was detected by molecular docking. Finally, Immunohistochemistry (IHC) and TUNEL staining were used to verify the mechanism of XHXY and GPS in anti-GD. ResultsXHXY and GPS alleviated GD by ameliorating the pathological changes and reducing thyroxine and TRAb levels. In mouse serum, a total of 31 prototypical XHXY ingredients were detected, and the majority of these components were from monarch and minister medicine. Proteomics study results indicated that the XHXY may mainly regulate targets including FAS-associated death domain protein (FADD), Apolipoprotein C-III, etc. and main pathways are Apoptosis, Cholesterol metabolism, TNF signalling pathway, etc. Strong binding activity of the prototypical active ingredient and GPS towards FADD, Caspase 8, and Caspase 3 was demonstrated by molecular docking. XHXY and its primary component, GPS, elevated the expression of FADD, Caspase 8, and Caspase 3, and enhance apoptosis in thyroid cells, as lastly validated by TUNEL and IHC staining. ConclusionsXHXY exhibits a favorable therapeutic effect in treating GD by promoting apoptosis in thyroid cells through the upregulation of FADD, Caspase 8, and Caspase 3 expression. And GPS is the main efficacy material basis for its therapeutic effect in anti-GD.

Analysis of the phytochemical components of Prunella vulgaris using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry combined with molecular networking and assessment of their antioxidant and anti-α-glucosidase activities.

Prunella vulgaris has long been used in traditional medicine and is consumed as a tea in China. Here, the total phenolic and flavonoid concentrations of plants from different geographical regions were measured. It was found that the total phenolic acid concentration ranged from 4.15 to 8.82 g of gallic acid equivalent per 100 g of dry weight (DW), and the total flavonoid concentration was 4.67-7.33 g of rutin equivalent per 100 g DW. Antioxidant activities were measured using 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and the results ranged from 73.47% to 94.43% and 74.54% to 93.39%, respectively, whereas α-glucosidase inhibition was between 75.31% and 95.49%. Correlation analysis showed that the total flavonoids in P. vulgaris had superior antioxidant and anti-α-glucosidase activities compared to the total phenolic compounds. The active components of P. vulgaris were analyzed using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry combined with both classical molecular networking and feature-based molecular networking on the Global Natural Products Social platform, identifying 32 compounds, namely 14 flavonoids, 12 phenolic compounds, and 6 other chemical components. These results could provide useful information on the use of P. vulgaris as a functional tea.

Identification, Quantitation, and Sensory Evaluation of Thiols in Bordeaux Red Wine with Characteristic Aging Bouquet.

Great Bordeaux red wines are known for their distinctive aging bouquet. However, the nature of volatile chemicals underpinning this sensory expression is not fully understood. This work investigated the empyreumatic aging bouquet of a collection of premium Bordeaux red wines using silver-ion (Ag+) solid-phase extraction, cryogenic heart-cutting multidimensional gas chromatography mass spectrometry/olfactometry, and comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry. In doing so, a substantial number of "meaty" odors were revealed. Three detected "meaty" notes were tentatively or unequivocally attributed to furan thiols. Among them, 2-methyltetrahydrofuran-3-thiol (1) with a pleasant "meaty" aroma was reported in wine for the first time. Its trans isomer (trans-1a) was resolved from its racemate by chemical modification, which confirmed its presence in wine. The odor detection threshold of trans-1a in the model wine was determined at 55 ng/L. Moreover, an additive effect between 1 and literature-known 2-methyl-3-furanthiol was observed. By a new ultra high-performance liquid chromatography quadrupole Orbitrap high-resolution mass spectrometry method, the concentration of trans-1a, in addition to those of 2-methyl-3-furanthiol and 2-furfuryl thiol, was measured in the wines at ng/L levels.

The effect of polyphenols on DNA methylation-assessed biological age attenuation: the DIRECT PLUS randomized controlled trial

BackgroundEpigenetic age is an estimator of biological age based on DNA methylation; its discrepancy from chronologic age warrants further investigation. We recently reported that greater polyphenol intake benefitted ectopic fats, brain function, and gut microbiota profile, corresponding with elevated urine polyphenols. The effect of polyphenol-rich dietary interventions on biological aging is yet to be determined.MethodsWe calculated different biological aging epigenetic clocks of different generations (Horvath2013, Hannum2013, Li2018, Horvath skin and blood2018, PhenoAge2018, PCGrimAge2022), their corresponding age and intrinsic age accelerations, and DunedinPACE, all based on DNA methylation (Illumina EPIC array; pre-specified secondary outcome) for 256 participants with abdominal obesity or dyslipidemia, before and after the 18-month DIRECT PLUS randomized controlled trial. Three interventions were assigned: healthy dietary guidelines, a Mediterranean (MED) diet, and a polyphenol-rich, low-red/processed meat Green-MED diet. Both MED groups consumed 28 g walnuts/day (+ 440 mg/day polyphenols). The Green-MED group consumed green tea (3–4 cups/day) and Mankai (Wolffia globosa strain) 500-ml green shake (+ 800 mg/day polyphenols). Adherence to the Green-MED diet was assessed by questionnaire and urine polyphenols metabolomics (high-performance liquid chromatography quadrupole time of flight).ResultsBaseline chronological age (51.3 ± 10.6 years) was significantly correlated with all methylation age (mAge) clocks with correlations ranging from 0.83 to 0.95; p < 2.2e − 16 for all. While all interventions did not differ in terms of changes between mAge clocks, greater Green-Med diet adherence was associated with a lower 18-month relative change (i.e., greater mAge attenuation) in Li and Hannum mAge (beta = − 0.41, p = 0.004 and beta = − 0.38, p = 0.03, respectively; multivariate models). Greater Li mAge attenuation (multivariate models adjusted for age, sex, baseline mAge, and weight loss) was mostly affected by higher intake of Mankai (beta = − 1.8; p = 0.061) and green tea (beta = − 1.57; p = 0.0016) and corresponded with elevated urine polyphenols: hydroxytyrosol, tyrosol, and urolithin C (p < 0.05 for all) and urolithin A (p = 0.08), highly common in green plants. Overall, participants undergoing either MED-style diet had ~ 8.9 months favorable difference between the observed and expected Li mAge at the end of the intervention (p = 0.02).ConclusionsThis study showed that MED and green-MED diets with increased polyphenols intake, such as green tea and Mankai, are inversely associated with biological aging. To the best of our knowledge, this is the first clinical trial to indicate a potential link between polyphenol intake, urine polyphenols, and biological aging.Trial registrationClinicalTrials.gov, NCT03020186.

Novel Isotope Dilution High-Performance Liquid Chromatography Quadrupole–Linear Ion Trap Tandem Mass Spectrometry Method for the Determination of Five Steroid Hormones in Human Serum

Accurate measurement of endogenous steroid hormones in serum is a powerful tool for understanding the status of human adrenal diseases. Isotope dilution–liquid chromatography–tandem mass spectrometry is the most commonly used method for determination of steroids in serum. In this study, we used a laboratory-constructed quadrupole-linear ion trap (Q-LIT) tandem mass spectrometer to develop a new method for the quantification of five steroid hormones in serum. A simple and efficient pretreatment was improved, wherein the serum was extracted once by a mixed solvent of 1.0 mL of ethyl acetate and n-hexane. Using the optimized method, the regression coefficients of the calibration curves were all higher than 0.99, the limits of detection were from 0.31 to 1.25 ng mL−1, and the limits of quantification were between 1.00 and 2.50 ng mL−1. The intra-day (n = 3) relative standard deviations ranged from 4.24% to 13.79%, and the inter-day (n = 3) relative standard deviations from 3.99% to 11.43%. After internal standard correction, the relative recoveries of all steroids were from 86.08% to 106.60%. Twenty-two serum samples were analyzed by Q-LIT, which was compared with commercial triple quadrupole mass spectrometer. The results of the instruments had high consistency. Therefore, Q-LIT accurately quantified steroid hormones in human samples and may be used for clinical diagnosis and monitoring of hormone-dependent diseases.

Uncovering the molecular mechanisms of Fructus Choerospondiatis against coronary heart disease using network pharmacology analysis and experimental pharmacology

Fructus Choerospondiatis (FC), a Mongolian medicine, was mainly used in Mongolian medical theory for the treatment of coronary heart disease (CHD). Nonetheless, the main components and mechanisms of action of FC in the treatment of coronary artery disease have not been studied clearly. Aim of the study: The aim of this study is to identify the components of FC and analyze the pathways affected by the targets of these components to probe into the potential mechanisms of action of FC on coronary heart disease. Materials and methods: Identification of compounds in FC employing high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF-MS) method, then further investigate the network pharmacology and molecular docking to obtain potential targets and elucidate the potential mechanism of action of FC in the therapy of CHD. Experimental validation was established to verify the mechanism of FC in vitro. Results: 21 FC components were identified and 65 overlapping targets were gained. In addition, these ingredients regulated AMPK and PPAR signaling pathway by 65 target genes including IL6, AKT1 and PPARg, etc. Molecular docking displayed that the binding ability of the key target PPARg to FC components turned out to be better. Experimental validation proved that FC treatment decreased the expression of PPARg (p < 0.05) compare with model group, which may be involved in the PPAR signaling pathway. Conclusions: This study was the first to elucidate the mechanism of action of components of FC for the treatment of CHD using network pharmacology. It alleviated CHD by inhibiting the expression of PPARg to attenuate hypoxia/reoxygenation injury, and the results give a basis for elucidating the molecular mechanism of action of FC for the treatment of coronary heart disease.

Flavonoid components and anti-photoaging activity of flower extracts from six Paeonia cultivars

Genus Paeonia is an important ornamental and medicinal crop. The flowers of Paeonia are rich in flavonoids, and flavonoids have excellent antioxidant and anti-inflammatory activities, which have the potential to slow down skin aging. In this study, flavonoids from whole flowers of six popular Paeonia cultivars were extracted using three different solvents. The composition and content of peony flowers flavonoids extracted with three solvents were determined by high performance liquid chromatography quadrupole time-of-flight mass spectrometry / mass spectrometry (HPLC-Q-TOF-MS/MS) and HPLC-diode array detection (DAD). A total of 26 flavonoids were isolated and identified, and 60 % ethanol showed the highest extraction efficiency. Not only flavonoids composition but also the content was significantly different in different peony flowers, among which ‘Hei Tian E’ had the most abundant composition (17 flavonoids) and the highest total content of flavonoids (57.75 mg/g DW by using 60 % ethanol). After that, the anti-skin photoaging effects of the 60 % ethanol extracts of the six Paeonia cultivars flowers were evaluated by UVR-Fulkutis and UVA-Fibroblasts photoaging models. Under exposure to UV radiation, some Paeonia flowers’ extracts can prevent the decrease of skin surface thickness and reduce the loss of Collagen IV and VII. In addition, the production and accumulation of ROS, MMP-1 and MMP-3 in fibroblasts were inhibited, indicating the attenuation of skin photoaging by the extracts. Among the six samples tested, ‘Hei Tian E’ whole flower extract (BH-HT) had the best comprehensive effect. Finally, the correlations between components and anti-photoaging efficacy were analyzed. We speculate that the anti-photoaging effect was significantly related to its richness in quercetin 3-O-di-glucoside, quercetin 3-O-glucoside, isorhamnetin 3-O-galloyl arabinoside, cyanidin 3,5-di-O-glucoside, cyanidin 3-O-glucoside, and peonidin 3-O-glucoside. In general, the results show that Paeonia flowers have potential for development of anti-photoaging herbal essence products.

Oral Ingestion of AP Collagen Peptide Leads to Systemic Absorption of Gly-Pro-Hyp, Alleviating H2O2-Induced Dermal Fibroblast Aging

Collagen-derived dipeptides and tripeptides have various physiological activities. In this study, we compared the plasma kinetics of free Hyp, peptide-derived Hyp, Pro-Hyp, cyclo(Pro-Hyp), Hyp-Gly, Gly-Pro-Hyp, and Gly-Pro-Ala after ingestion of four different collagen samples: AP collagen peptide (APCP), general collagen peptide, collagen, and APCP and γ-aminobutyric acid (GABA) combination. Each peptide was measured by high-performance liquid chromatography and triple quadrupole mass spectrometer. We found that, among all the peptides that were analyzed, only Gly-Pro-Hyp was significantly increased after ingestion of APCP compared with that of general collagen peptides and collagen. In addition, ingestion of the APCP and GABA combination improved the absorption efficiency of Gly-Pro-Ala. Finally, we reveal that Gly-Pro-Hyp was effective for preventing H2O2-induced reduction in extracellular matrix (ECM)-related genes, COL1A, elastin, and fibronectin, in dermal fibroblasts. Taken together, APCP significantly enhances the absorption of Gly-Pro-Hyp, which might act as an ECM-associated signaling factor in dermal fibroblasts, and the APCP and GABA combination promotes Gly-Pro-Ala absorption. Clinical Trial Registration number: UMIN000047972.

Aspartame-Sweetened Tap Water: Transformation Products and 2,6-Dichloro-1,4-Benzoquinone Formation.

Aspartame (APM), a dipeptide of aspartic acid (ASP) and phenylalanine (PHE), is a widely used artificial sweetener in beverages. It is unclear whether residual chlorine in tap water can react with APM to form disinfection byproducts (DBPs). Therefore, we investigated the formation of DBPs from the reaction of APM with residual chlorine in authentic tap water. APM and a commercial sweetener (CS) packet containing APM were studied under authentic and simulated tap water conditions. Eight chlorinated products of APM were detected using solid-phase extraction (SPE) and high performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS). These new chloro-products were tentatively identified based on accurate masses, isotopic patterns of 35,37Cl, and MS/MS spectra. Furthermore, we identified APM as a precursor to 2,6-dichloro-1,4-benzoquinone (DCBQ). DCBQ significantly increased to 2.3-12 ng/L with the addition of APM or CS in tap waters collected from different locations compared to 1.4-1.8 ng/L in the same tap water samples without sweetener. DCBQ and two of the chlorinated transformation products were identified in cold prepared tea containing APM. DCBQ formation was eliminated when the residual chlorine in tap water was reduced by ascorbic acid or boiling prior to the addition of APM or CS. This study found that eight new DBPs and DCBQ were produced by the reactions of residual chlorine with APM and CS. These findings show an unintended exposure source of emerging DBPs via APM sweetened beverages.

Identification of Novel Parishin Compounds from the Twig of Maclura tricuspidata and Comparative Analysis of Parishin Derivatives in Different Parts

Parishin compounds are rare polyphenolic glucosides mainly found in the rhizome of the traditional Chinese medicinal plant, Gastrodia elata. These constituents are reported to have several biological and pharmacological activities. In the present study, two novel parishin derivatives not previously reported as plant-based phytochemicals were identified from a twig of Maclura tricuspidata (MT) and two new compounds were elucidated as 1-(4-(β-d-glucopyranosyloxy)benzyl)-3-hydroxy-3-methylpentane-1,5-dioate (named macluraparishin E) and 1,3-bis(4-(β-d-glucopyranosyloxy)benzyl)-3-hydroxy-3-methylpentane- 1,5-dioate (macluraparishin C), based on the experimental data obtained by UV-Visible (UV-Vis) spectroscopy, high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) and nuclear magnetic resonance (NMR) spectroscopy. Additionally, gastrodin, parishin A and parishin B were positively identified by spectroscopic evidence and the comparison of HPLC retention time with the corresponding authentic standards. Gastrodin, parishin A and parishin B, macluraparishin E and macluraparishin C were found to be the most abundant constituents in the MT twig. The compositions and contents of these constituents were found to vary depending on the different parts of the MT plant. In particular, the contents of parishin A, parishin B, macluraparishin C and macluraparishin E were higher in the twig, bark and root than in the leaves, xylem and fruit.

Analysis of 12 Chemical Compounds And Pattern Recognition of Different Parts of Angelicae Sinensis Radix by Liquid Chromatography-Tandem Mass Spectrometry And Chemometrics Methods.

To evaluate the quality and quantify bioactive constituents in different parts of Angelicae Sinensis Radix, an efficient, high-speed, high-sensitivity high-performance liquid chromatography and triple quadrupole mass spectrometry method was used for simultaneous detection of 12 chemical compounds including L-tryptophan, chlorogenic acid, caffeic acid, ferulic acid, isoferulic acid, senkyunolide I, guanosine, proline, L-glutamine, γ-aminobutyric acid, glutamic acid, and arginine in 52 batches of Angelicae Sinensis Radix from Gansu, China. The established methods were validated by good linearity (R2≥0.9921), limits of detection (0.0001-0.0156 μg/mL), limits of quantitation (0.0006-0.0781 μg/mL), stability (RSD≤7.77%), repeatability (RSD≤6.79%), intra- and interday precisions (RSD≤6.00% and RSD≤6.39%, respectively) and recovery (90.90-107.16%). According to the quantitative results, the contents of the hydrophilic compounds were higher in the head, while the medium and weak polar components were mainly concentrated in the tail. Finally, principal component analysis results revealed that Angelicae Sinensis Radix could be divided into different medicinal sites based on polar components such as amino acids, nucleosides. The combination of liquid chromatography-tandem mass spectrometry and principal component analysis is a simple and reliable method for pattern recognition and quality evaluation of Angelicae Sinensis Radix.

Optimization of Cordyceps sinensis fermentation Marsdenia tenacissima process and the differences of metabolites before and after fermentation

In this paper, we explored the interaction of factors which influenced the Cordyceps sinensis fermentation Marsdenia tenacissima (Roxb.) Wight et Arn, a Dai (a national minority of China) medicine, and the optimal fermentation conditions. The differences of C. sinensis metabolites in normal state (CN) and products of two-way liquid fermentation of C. sinensis and Marsdenia tenacissima (CM) and Marsdenia tenacissima (MT). The interactive effect of factors was analyzed and the best conditions are obtained through the box-behnken design (BBD) in response surface methodology (RSM). All metabolites were determined by ultra high performance liquid chromatography quadrupole time of flight mass spectrometer (UHPLC-Q-TOF-MS), analyzed and identified by metabonomics technology. Results showed that the optimum fermentation conditions were the concentration of raw medicinal materials is 160 g/L, the fermentation time is 6 days, the inoculation volume is 9.5%, the rotating speed is 170 rpm. 197 metabolites were identified in both positive ion and negative ion. 119 metabolites were significantly different between CN and CM. 43 metabolites were significantly different between CM and MT. Differential metabolic pathways were enriched. In conclusion, this paper optimizes the bidirectional fermentation process of M. tenacissima and C. sinensis through response surface methodology, and analyzes the changes of components from the level of metabonomics, so as to provide reference for exploring medicinal fungi fermentation of traditional Chinese medicine.

Structure Analysis of Unsaturated Polymyxin E Components Based on High-Performance Liquid Chromatography - Quadrupole/ Time of Flight Tandem Mass Spectrometry and Photochemical Reaction

Background: Polymyxin E (PME), which is a complex of cationic cyclic lipodecapeptides, is used to treat multidrug-resistant gram-negative bacterial infections. Besides the main components PME1 and PME2, polymyxin containing unsaturated fatty acyl (FA) group with lower contents is hardly to determine the structure without chromatographic preparations and NMR. Introduction: The peptide sequences of PME components has been carried out based on high performance liquid chromatography-quadrupole / time-of-flight mass spectrometry (HPLC-Q/TOF-MS). However, the components with double bond on the FA, such as 2’, 3’-dehydro PME1, were difficult to be determined or easily misjudged by MS/MS. The transformation of such unsaturated components to be epoxidized components or di-hydroxylated components can promote the acquisition of more fragment ions in the MS/MS, so as to assist in judging the position of double bonds on FA. Methods: In this paper, the PME mixtures were dissolved in an equal proportion of 20% ACN aqueous solution and 2-acetylpyridine. The above PME solution was transferred to a quartz cuvette and irradiated with the ultraviolet lamp at 254 nm for 8h. The dehydro PME components were converted to be epoxy PMEs and dihydroxy PMEs. A fragmentation pathway of epoxidized components or di-hydroxylated components based on Q/TOF-MS/MS was proposed for the first time. Results: According to the characteristic ions of epoxidized components and di-hydroxylated components, 2’, 3’-epoxy PME1/E2 and 2’, 3’-dihydroxy PME1/E2 were confirmed. It can be inferred that the double bond is located at the 2’, 3’-position of FA. Conclusion: The structure of unsaturated PME component with double bond on the FA is elucidated by HPLC-Q/TOF-MS combined with photochemical reaction. This strategy is applicable to other lipopeptides containing unsaturated FA chain.

Antimicrobial Activity of Smilax china L. Root Extracts against the Acne-Causing Bacterium, Cutibacterium acnes, and Its Active Compounds

The root of Smilax china L. is used in traditional Korean medicine. We found that the Smilax china L. root extract has strong antimicrobial activity against two Cutibacterium acnes strains (KCTC 3314 and KCTC 3320). The aim of this study was to identify the beneficial properties of Smilax china L. extracts for their potential use as active ingredients in cosmetics for the treatment of human skin acne. The high-performance liquid chromatography (HPLC) and liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC/QTOF/MS) methods were used to obtain the profile of secondary metabolites from the ethyl acetate-soluble fraction of the crude extract. Agar diffusion and resazurin-based broth microdilution assays were used to evaluate antimicrobial activity and minimum inhibitory concentrations (MIC), respectively. Among the 24 metabolites, quercetin, resveratrol, and oxyresveratrol were the most potent compounds against Cutibacterium acnes. Minimum inhibitory concentrations of quercetin, resveratrol, and oxyresveratrol were 31.25, 125, and 250 μg/mL, respectively.

Understanding the metabolism of the novel plant antiviral agent dufulin by different positional 14C labeling in cherry radishes

Dufulin is a new type of plant antiviral agent. However, its metabolism in plants, which is very important for environmental risk assessment, is still unclear. In this study, we used 14C markers at different positions and high-performance liquid chromatography–quadrupole time-of-flight–mass spectrometry (HPLC–QTOF–MS) to qualitatively and quantitatively analyze dufulin metabolites in cherry radish. By combining ion pairs with unique abundance ratios, we clarified the metabolite structures, inferred the metabolic pathway of dufulin, and clarified the criteria for residues. The extractable residue of dufulin from cherry radish stem and leaf tissues was above 98 % and that in the succulent root was above 87 %. In the stem and leaf tissues and succulent root, dufulin underwent both phase I and phase II metabolism, and four metabolites were produced, including a conjugate of glucose malonate and hydroxylated dufulin, which was confirmed by comparison with a standard. However, the proportions and concentrations of the four metabolites did not meet the residue criteria, so only the dufulin precursor compound was included as a residue. This study provides reliable data for evaluating the impacts of dufulin on the environment and human health and for objectively examining the safety of dufulin.

Xiehuo Xiaoying decoction inhibits Tfh cell expansion and promotes Tfr cell amplification to ameliorate Graves’ disease

Ethnopharmacological relevanceXiehuo Xiaoying decoction (XHXY) has shown great potential in the treatment of GD, but its mechanism remains obscure. Increase of follicular helper T (Tfh) cells and reduction of follicular regulatory T (Tfr) cells contribute to a high thyrotropin receptor antibodies (TRAb) level and possible Graves’ disease (GD). Oxidative stress (OS) disrupts T helper cell differentiation and aggravates autoimmunity. Aim of the studyThis study aimed to investigate whether XHXY decoction can ameliorate autoimmunity in GD via inhibiting OS and regulating Tfh and Tfr cells. Materials and methodsThe main XHXY bioactive compounds were identified using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. GD was induced in the mice through three intramuscular injections of adenovirus expressing the TSH receptor. Then, the mice received oral gavage of XHXY (17 g/kg·d) and 34 g/kg·d) for 4 weeks. OS indicators were assessed. Flow cytometry was used to confirm the proportion of Tfh and Tfr cells in the lymph nodes and spleens of the mice. Cytokine expression levels were determined using enzyme-linked immunosorbent assay. Factors including interleukin-21, B-cell lymphoma-6, and forkhead box P3 (Foxp3) were detected using quantitative polymerase chain reaction. The mRNA and protein expression levels of Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid-2-related factor 2 (Nrf2), and haem oxygenase 1 (HO-1) were detected using quantitative polymerase chain reaction and Western blotting, respectively. ResultsTwelve main ingredients of XHXY were identified. XHXY relieved GD by lowering thyroxine (p < 0.01) and TRAb levels (p < 0.01). XHXY ameliorated OS by decreasing the levels of NADPH oxidase 2 (p < 0.05), 4-hydroxynonenal (p < 0.01), and 8-oxo-2′-deoxyguanosine (p < 0.001). It inhibited Tfh cell expansion (p < 0.05), as well as the production of cytokine interleukin −21 (p < 0.01), interleukin −4 (p < 0.01) and transcription factor B-cell lymphoma 6 (p < 0.05). XHXY also induced Tfr cell amplification (p < 0.05), increased the production of interleukin −10 (p < 0.05) and transforming growth factor β (p < 0.05) and the mRNA levels of Foxp3 (p < 0.05). Finally, the Tfh/Tfr ratio returned to normal. In addition, XHXY activated Nrf2 and HO-1 expression, but inhibited Keap1 activation. ConclusionsXHXY relieves autoimmunity in GD via inhibiting Tfh cell amplification and Tfr cell reduction, a mechanism which probably involves the Keap1/Nrf2 signaling pathway.