MS Method Research Articles

Comparison of APCI orbitrap MS and GCxGC/EI TOF MS for the hydrocarbon analysis of heavy base oils

The performance of positive-ion mode atmospheric pressure chemical ionization linear quadrupole ion trap orbitrap mass spectrometry (APCI LQIT Orbitrap MS) has been compared to that of two-dimensional gas chromatography coupled with positive-ion mode electron ionization time-of-flight mass spectrometry (GCxGC/EI TOF MS) for the analysis of group III heavy base oils. Upon APCI, all compounds were predominantly ionized via protonation. However, protonated alkanes are not stable and predominantly generated [M−H]+ ions via elimination of a hydrogen molecule from the protonated molecule. The high-resolution APCI mass spectra provided elemental composition information for the compounds while low-resolution APCI mass spectra were used for semiquantitative evaluation of the relative amounts of the compounds. Ions corresponding to compounds in seven hydrocarbon classes, i.e., (1) linear and branched alkanes, (2) monocycloalkanes, (3) dicycloalkanes, (4) tricycloalkanes and alkylbenzenes, (5) tetracycloalkanes, (6) pentacycloalkanes, and (7) hexacycloalkanes were identified. On the other hand, the GCxGC/EI TOF MS method enabled the chromatographic separation of most analytes. The measured EI mass spectra were used for the identification of the compounds while the GC peak areas were used for semiquantitative evaluation. The detected compounds were classified into three different hydrocarbon classes, (1) linear and branched alkanes, (2) cycloalkanes, and (3) aromatic hydrocarbons. The (+)APCI LQIT Orbitrap MS method was found to be faster and to provide more information for the larger compounds in the samples. For example, polycycloalkanes were only detected by using (+)APCI MS. However, no aromatic compounds were detected. Data processing was more straightforward than when using the GCxGC/EI TOF MS method. However, the latter method provided more accurate results for aromatic hydrocarbons and small alkanes as the analysis of small alkanes by using (+)APCI MS is limited due to the fact that [M−H]+ ions of small alkanes overlap with alkane fragment ions. Both light and heavy aliphatic and aromatic hydrocarbons were detected by using the GCxGC/EI TOF MS method but not polycycloalkanes. Therefore, these two methods complement each other, and each has different strengths and weaknesses.

In-Depth Excavation and Screening of Triterpene Saponins From the Rhizome of Panax japonicus Using High Performance Liquid Chromatography Coupled to Electrospray Ionization and Quadrupole Time-of-Flight Mass Spectrometry.

The rhizome of Panax japonicus (RPJ) has been used for thousands of years in west China. Triterpene saponins (TSs) were considered to be the main pharmacologically bioactive ingredients in RPJ. However, it is difficult and time-consuming to profile and identify them according to the traditional phytochemical methods. High-performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS/MS) was used for chemical identification of TSs from the extract of RPJ in negative ion mode. Their chemical structures were tentatively elucidated based on exact formulas, fragmentation patterns and literature data. In all, 42 TSs were discovered and tentatively characterized in RPJ, of which 12 TSs were identified as potential new compounds according to their molecular mass, fragmentation pattern and chromatographic behavior. The results demonstrated that the developed HPLC-ESI-QTOF-MS/MS method was conducive to the discovery of the active ingredients of RPJ and the establishment of quality standards.

Enterokokların MALDI-TOF MS’de Slanetz ve Bartley ve Kanlı Agar Besiyeri ile Karşılaştırılarak Tanımlanması

Introduction: Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used frequently in the last decade as a fast and reliable method for the identification of microorganisms. The fact that only one gram-negative selective medium is recommended in this method causes problems in the diagnosis. There is no information in the scientific literature that Slanetz and Bartley (SB) agar used in the study can be used in MALDI-TOF MS to identify bacteria. Materials and Methods: In this study, it was aimed to compare the identification performance of enterococci isolated from drinking-use and lake waters in Van and its surroundings with blood agar by using MALDI-TOF MS method and to develop a simple and fast sample preparation method that increases this performance. For this purpose, 60 mains and 30 lake water samples were examined for six months. Results: The samples were studied by the membrane filtration method. The isolated enterococci were identified by MALDI-TOF MS method with the isolates taken directly from both blood agar and SB medium, which is a selective medium. The same results were obtained in the species determination in both media. According to these results, 35% of the mains water isolates were Enterococcus casseliflavus, 30% Enterococcus faecalis, 26.7% Enterococcus faecium, 6.6% Enterococcus hirae, 1.7% Enterococcus columbae, and 20% of lake water isolates E. casseliflavus, 40% E. faecalis, 36.7% E. faecium, 3.3% E. hirae, E. casseliflavus in mains water and E. faecalis in lake water are the most frequently isolated enterococci species. Conclusion: Determining that it can also be obtained from selective media such as SB agar, for which there is no data on its suitability for mass spectrometric identification, will contribute to the more effective and rapid use of MALDI-TOF MS in microbiology laboratories. In addition, it has been concluded that all the waters examined are exposed to a significant level of microbial contamination and pose a potential danger to public health. It has been revealed that the consumption of these waters may pose a potential health risk for immunocompromised patients and the elderly, as well as children.

A multiplex method for detection of SARS-CoV-2 variants based on MALDI-TOF mass spectrometry

The recent outbreak of the coronavirus disease 2019 (COVID-19) pandemic and the continuous evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have highlighted the significance of new detection methods for global monitoring and prevention. Although quantitative reverse transcription PCR (RT-qPCR), the current gold standard for diagnosis, performs excellently in genetic testing, its multiplexing capability is limited because of the signal crosstalk of various fluorophores. Herein, we present a highly efficient platform which combines 17-plex assays with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), enabling the targeting of 14 different mutation sites of the spike gene. Diagnosis using a set of 324 nasopharyngeal swabs or sputum clinical samples with SARS-CoV-2 MS method was identical to that with the RT-qPCR. The detection consistency of mutation sites was 97.9% (47/48) compared to Sanger sequencing without cross-reaction with other respiratory-related pathogens. Therefore, the MS method is highly potent to track and assess SARS-CoV-2 changes in a timely manner, thereby aiding the continuous response to viral variation and prevention of further transmission.

Association of eosinophil-derived neurotoxin levels with asthma control status in patients with aspirin-exacerbated respiratory disease.

The long-term goals of asthma treatment are to achieve well control of symptoms and to minimize the future risk of asthma exacerbation. Identifying biomarkers for uncontrolled asthma is important for improving the asthma outcome. This study aimed to investigate the association of the levels of eosinophil-derived neurotoxin (EDN) with asthma control status in specific asthma phenotype, aspirin-exacerbated respiratory disease (AERD), and aspirin-tolerant asthma (ATA). A total of 136 adult asthmatics, including 47 asthmatics with AERD and 89 asthmatics with ATA, were enrolled. Plasma, sputum, and urine were collected at enrollment and the levels of EDN were measured by the K-EDN ELISA kit. Urinary leukotriene E4 (LTE4 ) level was measured using liquid chromatography-mass spectrometry (LC-MS)/MS methods. Asthma control status was evaluated according to the GINA guideline, asthma control test and asthma control questionnaire scores. In the total study subjects, sputum levels of EDN as well as of urine and plasma EDN showed significantly higher levels in patients with uncontrolled asthma than in those with well-controlled or partly-controlled asthma (ANOVA, p<0.001); in patients with AERD, the sputum EDN levels showed significant correlations with ACT, ACQ, and AQLQ scores (p=0.010, r=-0.536, p=0.001, r=0.665, and p<0.001, r=-0.691, respectively), while no differences were noted in patients with ATA. Sputum EDN level was the only significant factor for ACT, ACQ, and AQLQ scores in patients with AERD (p=0.001, p<0.001, and p<0.001, respectively) in the multivariate analysis adjusting for age, sex, peripheral eosinophil count, and urine LTE4 . The ROC curve analysis demonstrated that sputum EDN can predict uncontrolled asthma with 80% sensitivity and 88.2% specificity for ACT≤19 (area under the ROC curve [AUC]=0.824, p=0.019); 71.4% sensitivity and 86.7% specificity for ACQ≥1.5 (AUC=0.752, p=0.049) only in AERD patients. The level of sputum EDN may be a potential biomarker for identifying the asthma control status in patients with AERD.

Quantitative analysis of ethanolamine plasmalogen species in red blood cells using liquid chromatography tandem mass spectrometry for diagnosing peroxisome biogenesis disorders

Plasmalogens are glycerophospholipids characterized by a vinyl-ether bond with a fatty alcohol at the sn-1 position, a polyunsaturated fatty acid at the sn-2 position, and a polar head at the sn-3 position, commonly phosphoethanolamine. Plasmalogens play crucial roles in several cellular processes. Reduced levels have been associated with Alzheimer’s and Parkinson’s disease progression. Markedly reduced plasmalogens are a classic feature of peroxisome biogenesis disorders (PBD) because plasmalogen synthesis requires functional peroxisomes. Particularly, severe plasmalogen deficiency is the biochemical hallmark of rhizomelic chondrodysplasia punctata (RCDP). Traditionally, plasmalogens are evaluated in red blood cells (RBCs) by gas-chromatography/mass-spectrometry (GC–MS), which cannot distinguish individual species. We developed a liquid-chromatography/tandem mass-spectrometry (LC-MS/MS) method to quantify eighteen phosphoethanolamine plasmalogens in RBCs to diagnose PBD patients, especially RCDP. Validation results showed a specific, robust, and precise method with broad analytical range. Age-specific reference intervals were established; control medians were used to assess plasmalogen deficiency in patients’ RBCs. Clinical utility was also confirmed in Pex7 deficient mouse models recapitulating severe and milder RCDP clinical phenotypes. To our knowledge, this is the first attempt to replace the GC–MS method in the clinical laboratory. In addition to diagnosing PBDs, structure-specific plasmalogen quantitation could help understand disease pathogenesis and monitor therapy.

Development of a Fully Automated Method HS-SPME-GC-MS/MS for the Determination of Odor-Active Carbonyls in Wines: a "Green" Approach to Improve Robustness and Productivity in the Oenological Analytical Chemistry.

The aim of this study was the optimization and validation of a green, robust, and comprehensive method for the determination of volatile carbonyl compounds (VCCs) in wines that could be added as a new quality control tool for the evaluation of a complete fermentation, correct winemaking style, and proper bottling and storage. A HS-SPME-GC-MS/MS method was optimized and automated using the autosampler to improve overall performance. A solvent-less technique and a strong minimization of all volumes were implemented to comply with the green analytical chemistry principles. There were as many as 44 VCC (mainly linear aldehydes, Strecker aldehydes, unsaturated aldehydes, ketones, and many other) analytes under investigation. All compounds showed a good linearity, and the LOQs were abundantly under the relevant perception thresholds. Intraday, 5-day interday repeatability, and recovery performances in a spiked real sample were evaluated showing satisfactory results. The method was applied to determine the evolution of VCCs in white and red wines after accelerated aging for 5 weeks at 50 °C. Furans and linear and Strecker aldehydes were the compounds that showed the most important variation; many VCCs increased in both classes of samples, whereas some showed different behaviors between white and red cultivars. The obtained results are in strong accordance with the latest models on carbonyl evolution related to wine aging.

Design of Peptide Ligand for Lactoferrin and Study of Its Binding Specificity

The in silico modelling of peptides complementary to lactoferrin was carried out using the Protein 3D software package and replication of the natural bonding site between pneumococcal surface protein (PSP) and lactoferrin (LF). The modeling was based on analysis of the conjugated ion–hydrogen bond systems between these proteins (CIHBS). The oligopeptide EEVAPQAQAKIAELENQVHRLE was proposed via computer modelling and synthesized using the solid phase synthesis technique, purified, and analyzed with MS and HPLC methods to confirm &gt;95% purity. The peptide was then studied by capillary electrophoresis (CE). The CE experiments demonstrated the split of peptide zone in the presence of LF, due to complex formation and subsequent mobility change of the system peptide-protein. The reference experiments with homomyeloperoxidase and myoglobin did not show binding with LETI-11.

Phosphonylated tyrosine and lysine residues as biomarkers of local exposure of human hair to the organophosphorus nerve agents sarin and VX.

We herein present for the first time a micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS) procedure to detect phosphonylated tyrosine (Tyr) and lysine (Lys) residues obtained from human hair exposed to organophosphorus nerve agents (OPNA). In general, toxic OPNA react with endogenous blood proteins causing the formation of adducts representing well-known targets for biomedical analysis to prove exposure. In contrast, no protein-derived biomarker has been introduced so far to document local exposure of hair. Accordingly, we developed and characterized a μLC-ESI MS/HR MS method for the analysis of scalp hair exposed to OPNA in vitro. Type I and Type II keratin from hair was dissolved during lysis, precipitated and subjected to pronase-catalyzed hydrolysis yielding single adducted Lys and in a much higher amount Tyr residues. Exposure to sarin caused the adduction of an isopropyl methylphosphonic acid moiety and exposure to VX yielded adducts of ethyl methylphosphonic acid, well suited as biomarkers of exposure. These were of appropriate stability in the autosampler for 24 h. The biomarker yield obtained from hair of six individuals as well as from hair of six different parts of the body of one individual (armpit, beard, leg, arm, scalp, and pubic) differed reasonably indicating the variable individual protein composition and structure of hair. Exposed hair stored at ambient temperature for 9weeks with contact to air and daylight showed stability of all adducts and therefore their suitability for verification of exposure.

Investigation of the factors responsible for the low oral bioavailability of alizarin using a sensitive LC-MS/MS method: In vitro, in situ, and in vivo evaluations.

Alizarin (1,2-dihydroxyanthraquinone) is an anthraquinone reddish dye widely used for painting and textile dyeing. As the biological activity of alizarin has recently attracted increasing attention from researchers, its therapeutic potential as complementary and alternative medicine is of interest. However, no systematic research has been conducted on the biopharmaceutical and pharmacokinetic aspects of alizarin. Therefore, this study aimed to comprehensively investigate the oral absorption and intestinal/hepatic metabolism of alizarin using a simple and sensitive tandem mass spectrometry method developed and validated in-house. The present method for the bioanalysis of alizarin has merits, including a simple pretreatment procedure, small sample volume, and adequate sensitivity. Alizarin exhibited pH-dependent moderate lipophilicity and low solubility with limited intestinal luminal stability. Based on the in vivo pharmacokinetic data, the hepatic extraction ratio of alizarin was estimated to be 0.165-0.264, classified as a low level of hepatic extraction. In an in situ loop study, considerable fractions (28.2%-56.4%) of the alizarin dose were significantly absorbed in gut segments from the duodenum to ileum, suggesting that alizarin may be classified as the Biopharmaceutical Classification Systemclass II. An in vitro metabolism study using rat and human hepatic S9 fractions revealed that glucuronidation and sulfation, but not NADPH-mediated phase I reactions and methylation, are significantly involved in the hepatic metabolism of alizarin. Taken together, it can be estimated that the fractions of oral alizarin dose unabsorbed from the gut lumen and eliminated by the gut and liver before reaching the systemic circulation are 43.6%-76.7%, 0.474%-36.3%, and 3.77%-5.31% of the dose, respectively, resulting in a low oral bioavailability of 16.8%. Therefore, the oral bioavailability of alizarin depends primarily on its chemical degradation in the gut lumen and secondarily on first-pass metabolism.

Identification of Differential Compositions of Aqueous Extracts of Cinnamomi Ramulus and Cinnamomi Cortex

Cinnamomi ramulus (CR) and Cinnamomi cortex (CC), both sourced from Cinnamomum cassia Presl, are commonly used Chinese medicines in the Chinese Pharmacopeia. However, while CR functions to dissipate cold and to resolve external problems of the body, CC functions to warm the internal organs. To clarify the material basis of these different functions and clinical effects, a simple and reliable UPLC-Orbitrap-Exploris-120-MS/MS method combined with multivariate statistical analyses was established in this study with the aim of exploring the difference in chemical compositions of aqueous extracts of CR and CC. As the results indicated, a total of 58 compounds was identified, including nine flavonoids, 23 phenylpropanoids and phenolic acids, two coumarins, four lignans, four terpenoids, 11 organic acids and five other components. Of these compounds, 26 significant differential compounds were identified statistically including six unique components in CR and four unique components in CC. Additionally, a robust HPLC method combined with hierarchical clustering analysis (HCA) was developed to simultaneously determine the concentrations and differentiating capacities of five major active ingredients in CR and CC: coumarin, cinnamyl alcohol, cinnamic acid, 2-methoxycinnamic acid and cinnamaldehyde. The HCA results showed that these five components could be used as markers for successfully distinguishing CR and CC. Finally, molecular docking analyses were conducted to obtain the affinities between each of the abovementioned 26 differential components, focusing on targets involved in diabetes peripheral neuropathy (DPN). The results indicated that the special and high-concentration components in CR showed high docking scores of affinities with targets such as HbA1c and proteins in the AMPK-PGC1-SIRT3 signaling pathway, suggesting that CR has greater potential than CC for treating DPN.

Chemical constituents from leaves of Jatropha curcas

ObjectiveTo investigate the chemical constituents from the leaves of Jatropha curcas and evaluate their inhibition on lipopolysaccharide (LPS)-activated BV-2 microglia cells. MethodsThe n-BuOH extract of the leaves of J. curcas was isolated by macroporous adsorption resin, silica gel, ODS, column chromatography and semi-preparative HPLC. The structures of the compounds were identified by MS, NMR, ECD, and other spectroscopic methods. In addition, anti-neuroinflammatory effects of isolated compounds were evaluated by measuring the production of nitric oxide (NO) in over-activated BV-2 cells. ResultsSeventeen compounds, including (7R,8S)-crataegifin A-4-O-β-D-glucopyranoside (1), (8R,8′R)-arctigenin (2), arctigenin-4′-O-β-D-glucopyranoside (3), (-)-syringaresinol (4), syringaresinol-4′-O-β-D-glucopyranoside (5), (-)-pinoresinol (6), pinoresinol-4′-O-β-D-glucopyranoside (7), buddlenol D (8), (2R,3R)-dihydroquercetin (9), (2S,3S)-epicatechin (10), (2R,3S)-catechin (11), isovitexin (12), naringenin-7-O-β-D-glucopyranoside (13), chamaejasmin (14), neochamaejasmin B (15), isoneochamaejasmin A (16), and tomentin-5-O-β-D-glucopyranoside (17) were isolated and identified. Compounds 2, 4 and 8 significantly inhibited the release of NO in BV-2 microglia activated by LPS, with IC50 values of 18.34, 29.33 and 26.30 μmol/L, respectively. ConclusionCompound 1 is a novel compound, and compounds 2, 3, 8, 14–17 are isolated from Jatropha genus for the first time. In addition, the lignans significantly inhibited NO release and the inhibitory activity was decreased after glycosylation.

Refinement of a microfurnace pyrolysis-GC–MS method for quantification of tire and road wear particles (TRWP) in sediment and solid matrices

Tire and road wear particles (TRWP) are produced by abrasion at the interface of the pavement and tread surface and contain tread rubber with road mineral encrustations. Quantitative thermoanalytical methods capable of estimating TRWP concentrations are needed to assess the prevalence and environmental fate of these particles. However, the presence of complex organic constituents in sediment and other environmental samples presents a challenge to the reliable determination of TRWP concentrations using current pyrolysis-gas chromatography–mass spectrometry (Py-GC–MS) methodologies. We are unaware of a published study evaluating pretreatment and other method refinements for microfurnace Py-GC–MS analysis of the elastomeric polymers in TRWP including polymer-specific deuterated internal standards as specified in ISO Technical Specification (ISO/TS) 20593:2017 and ISO/TS 21396:2017. Thus, potential method refinements were evaluated for microfurnace Py-GC–MS, including chromatography parameter modification, chemical pretreatment, and thermal desorption for cryogenically-milled tire tread (CMTT) samples in an artificial sediment matrix and a sediment field sample. The tire tread dimer markers used for quantification were 4-vinylcyclohexene (4-VCH), a marker for styrene-butadiene rubber (SBR) and butadiene rubber (BR), 4-phenylcyclohexene (4-PCH), a marker for SBR, and dipentene (DP), a marker for natural rubber (NR) or isoprene. The resultant modifications included optimization of GC temperature and mass analyzer settings, along with sample pretreatment with potassium hydroxide (KOH) and thermal desorption. Peak resolution was improved while minimizing matrix interferences with overall accuracy and precision consistent with those typically observed in environmental sample analysis. The initial method detection limit for an artificial sediment matrix was approximately 180 mg/kg for a 10 mg sediment sample. A sediment and a retained suspended solids sample were also analyzed to illustrate the applicability of microfurnace Py-GC–MS towards complex environmental sample analysis. These refinements should help encourage the adoption of pyrolysis techniques for mass-based measurements of TRWP in environmental samples both near and distant from roadways.

Portable colorimetric enzymatic disposable biosensor for histamine and simultaneous histamine/tyramine determination using a smartphone

Tyramine oxidase (TAO), peroxidase (HRP), and Amplex Red (AR) have been immobilized on cellulose to obtain disposable biosensors for the determination of histamine. During the enzymatic reaction, AR is oxidized and a pink spot is obtained. Using a smartphone and measuring the G (green) color coordinate, histamine can be determined in the presence of other biogenic amines (putrescine and cadaverine) in concentrations ranging from 2·10−5 M to 5·10−4 M with a 7.5·10−6 M limit of detection (LoD). Despite tyramine interference, experimental conditions are provided which allow rapid and simple histamine and simultaneous histamine/tyramine (semi)quantitative determination in mixtures. Finally, tyramine and histamine were determined in a tuna extract with good results (compared to the reference HPLC–MS method). The methodology can also be applied in solution allowing histamine (and simultaneous histamine/tyramine) determination with a lower LoD (1.8·10−7 M) and a similar selectivity.Graphical abstract

Network pharmacology-based analysis of Resinacein S against non-alcoholic fatty liver disease by modulating lipid metabolism.

Ganoderma lucidum is reportedly the best source of traditional natural bioactive constituents. Ganoderma triterpenoids (GTs) have been verified as an alternative adjuvant for treating leukemia, cancer, hepatitis and diabetes. One of the major triterpenoids, Resinacein S, has been found to regulate lipid metabolism and mitochondrial biogenesis. Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease that has become a major public health problem. Given the regulatory effects on lipid metabolism of Resinacein S, we sought to explore potential protective effects against NAFLD. Resinacein S was extracted and isolated from G. lucidum. And mice were fed with high fat diet with or without Resinacein S to detect hepatic steatosis. According to Network Pharmacology and RNA-seq, we analyzed the hub genes of Resinacein S against NAFLD disease. Our results can be summarized as follows: (1) The structure of Resinacein S was elucidated using NMR and MS methods. (2) Resinacein S treatment could significantly attenuate high-fat diet (HFD)-induced hepatic steatosis and hepatic lipid accumulation in mouse. (3) GO terms, KEGG pathways and the PPI network of Resinacein S induced Differentially Expressed Genes (DEGs) demonstrated the key target genes of Resinacein S against NAFLD. (4) The hub proteins in PPI network analysis could be used for NAFLD diagnosis and treatment as drug targets. Resinacein S can significantly change the lipid metabolism in liver cells and yield a protective effect against steatosis and liver injury. Intersected proteins between NAFLD related genes and Resinacein S-induced DEGs, especially the hub protein in PPI network analysis, can be used to characterize targets of Resinacein S against NAFLD.

Bio-Guided Isolation of Compounds from Fraxinus excelsior Leaves with Anti-Inflammatory Activity.

Inflammation is the first physiological defence mechanism against external and internal stimuli. The prolonged or inappropriate response of the immune system may lead to the persistent inflammatory response that can potentially become a basis for chronic diseases e.g., asthma, type II diabetes or cancer. An important role in the alleviation of inflammatory processes, as an adjunct to traditional pharmacological therapy, is attributed to phytotherapy, especially to raw materials with a long tradition of use, e.g., ash leaves. Despite their long-term use in phytotherapy, the specific mechanisms of action have not been confirmed in a sufficient number of biological or clinical studies. The aim of the study is a detailed phytochemical analysis of infusion and its fractions, isolation of pure compounds from the leaves of Fraxinus excelsior and evaluation of their effect on the secretion of anti-inflammatory cytokines (TNF-α, IL-6) and IL-10 receptor expression in an in vitro model of monocyte/macrophage cells isolated from peripheral blood. Methods: Phytochemical analysis was carried out by the UHPLC-DAD-ESI-MS/MS method. Monocytes/macrophages were isolated from human peripheral blood using density gradient centrifugation on Pancoll. After 24 h incubation with tested fractions/subfractions and pure compounds, cells or their supernatants were studied, respectively, on IL-10 receptor expression by flow cytometry and IL-6, TNF-α, IL-1β secretion by the ELISA test. Results were presented with respect to Lipopolysaccharide (LPS) control and positive control with dexamethasone. Results: The infusion, 20% and 50% methanolic fractions and their subfractions, as well as their dominating compounds, e.g., ligstroside, formoside and oleoacteoside isolated from the leaves, show the ability to increase the IL-10 receptor expression on the surface of monocyte/macrophage cells, stimulated by LPS, and to decrease the secretion of pro-inflammatory cytokines, e.g., TNF-α, IL-6.

Potentiometric determination of mephedrone in oral fluids with ion‐selective membranes

AbstractThis work presents a potentiometric approach for the detection of two cathinone derivatives, namely mephedrone (4‐MMC) and clephedrone (4‐CMC). Contrary to neutral‐carriers (dibenzo‐18‐crown‐6‐ether and calix[4]arene), cation‐exchanger (tetraphenylborate sodium) as active component of ion‐selective membrane showed near‐Nernstian response (+47 mV/decade from 1.7 ⋅ 10−6 to 5.6 ⋅ 10−4 mol l−1) and selectivity for 4‐MMC in oral fluids (OF). The agreement between the quantitative results obtained with the potentiometric approach and the HPLC/MS‐MS method determination of 4‐MMC in OF shows the potential of the proposed potentiometric approach for forensic analysis.

Oral Wound Healing Potential of Polygoni Cuspidati Rhizoma et Radix Decoction-In Vitro Study.

Polygoni Cuspidati Rhizoma et Radix (syn. rhizomes of Reynoutria japonica Houtt.) is a pharmacopoeial raw material in Europe and China. In traditional medicine, one of the applications for Reynoutria japonica rhizomes is wound healing. In a recent in vitro study, we demonstrated that ethanol and acetone extracts from this herbal drug have the potential to heal oral gum wounds. However, considering that a majority of herbal medicines have been traditionally administered as water decoctions, in the present study, a decoction of Reynoutria japonica rhizomes was prepared and detailed tests to determine its in vitro gingival wound healing activity were conducted. We used the primary human gingival fibroblasts (HGF) incubated with a decoction to determine cell viability (MTT assay), cell proliferation (the confocal laser scanning microscope-CLSM), and cell migration (wound healing assay). Moreover, the collagen type III expression was examined using immunocytochemical staining. The studied decoction was qualitatively and quantitatively characterized using the validated HPLC/DAD/ESI-HR-QTOF-MS method. The Folin-Ciocalteu assay was used to determine the total phenols and tannins content. Additionally, HPLC-RI analysis of decoction and the previously obtained ethanol and acetone extracts was used to determine the composition of saccharides. Low concentration (from 50 to 1000 µg/mL) of decoction after 24 h incubation caused a significant increase in HGF cell viability. No cytotoxic effect was observed at any tested concentration (up to 2000 µg/mL). The lowest active concentration of decoction (50 µg/mL) was selected for further experiments. It significantly stimulated human gingival fibroblasts to proliferate, migrate, and increase the synthesis of collagen III. Phytochemical analysis showed significantly fewer polyphenols in the decoction than in the ethanol and acetone extracts tested earlier. In contrast, high levels of polysaccharides were observed. In our opinion, they may have a significant effect on the oral wound healing parameters analyzed in vitro. The results obtained encourage the use of this raw material in its traditional, safe form-decoction.

HPTLC–densitometric and HPTLC–MS Methods for Analysis of Triterpenoids from Cucurbita maxima Peel

Fruits and vegetables are integral part of our day to day. They are the main source of food for the living system. Large amount of agro-waste generated from food industries and kitchen pertaining to biggest nutritional loss, environmental problems and economical implications. Even though pomace, peels, rind and seeds are considered a waste part of plant they are still rich source of valuable compounds like polyphenols, oils, enzymes, vitamins, fiber, etc. which is just adjunct to its different uses. Present study conducted on waste pumpkin peel. HPTLC fingerprint profile of pumpkin peel methanol extract was performed using HPTLC plate of silica gel 60 F254 as the stationary phase and Toluene: Chloroform: Ethanol (4:4:1 V/V/V) as the mobile phase. After development chromatogram was scanned at 254 nm and 366 nm by densitometric scanner. Anisaldehyde in sulphuric acid is used as a detecting reagent which gives pink-violet and/or giving blue coloration after derivatization of plate. Further HPTLC-MS analysis gives the confirmation to detected terpenoids. Therefore, this waste peel part of the pumpkin can be used in an ingenious way by further purification, isolation and characterization.

A field trials-based study reveals the effects of chlormequat chloride on the quality of medicinal plant Salvia miltiorrhiza Bge. (Lamiaceae) in practical cultivation

The application of some specific plant growth regulators in the cultivation of herb medicines for yield promotion is a widespread phenomenon at present. But most of these agrochemical uses in medicinal plants are without effect verification and are illegal according to related regulations, thus it is necessary to conduct studies about the effect evaluation of these plant growth regulators on medicinal plants in actual production conditions. In this study, according to a preliminary investigation that chlormequat chloride (CCC) is widely used in the cultivation of the common herb Salvia miltiorrhiza, the effects of CCC on the quality of S. miltiorrhiza were comprehensively evaluated with both metabolomics-based UPLC/Q-TOF MS method and multi-component quantitative analysis based on samples from two years field trials. A global chemical constituent identification of S. miltiorrhiza was firstly carried out and consequently 47 tanshinones and 22 phenolics were identified or tentatively identified from the UPLC/Q-TOF MS data. Then, both the qualitative multivariate statistical analysis and quantitative determinations of major active components in S. miltiorrhiza samples that were treated with different dosages of CCC revealed that the application of CCC in S. miltiorrhiza cultivation is likely to count against the herbal quality and mainly manifested as the significant decrease of tanshinones content. Above all, this work provides a beneficial case for the recognition of the blind use of plant growth regulators in herb medicines for yield improvement and indicated that the use of PGRs in medicinal plant cultivation should be more cautious, and several further study points based on this work were discussed.