High Performance Liquid Chromatography-quadrupole Time Research Articles

Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutans.

Bacterial pathogens have remained a major public health concern for several decades. This study investigated the antibacterial activities of Miang extracts (at non-neutral and neutral pH) against Bacillus cereus TISTR 747, Escherichia coli ATCC 22595, Salmonella enterica serovar Typhimurium TISTR 292 and Streptococcus mutans DMST 18777. The potential of Polyvinylpolypyrrolidone (PVPP)-precipitated tannin-free Miang extracts in growth-inhibition of the cariogenic Streptococcus mutans DMST 18777 and its biofilms was also evaluated. The tannin-rich fermented extracts had the best bacterial growth inhibition against S. mutans DMST 18777 with an MIC of 0.29 and 0.72 mg/mL for nonfilamentous fungi (NFP) Miang and filamentous-fungi-processed (FFP) Miang respectively. This observed anti-streptococcal activity still remained after PVPP-mediated precipitation of bioactive tannins especially, in NFP and FFP Miang. Characterization of the PVPP-treated extracts using High performance liquid chromatography quadrupole-time of flight-mass spectrometry (HPLC-QToF-MS) analysis, also offered an insight into probable compound classes responsible for the activities. In addition, Crystal violet-staining also showed better IC50 values for NFP Miang (4.30 ± 0.66 mg/mL) and FFP Miang (12.73 ± 0.11 mg/mL) against S. mutans DMST 18777 biofilms in vitro. Homology modeling and molecular docking analysis using HPLC-MS identified ligands in tannin-free Miang supernatants, was performed against modelled S. mutans DMST 18777 sortase A enzyme. The in silico analysis suggested that the inhibition by NFP and FFP Miang might be attributed to the presence of ellagic acid, flavonoid aglycones, and glycosides. Thus, these Miang extracts could be optimized and explored as natural active pharmaceutical ingredients (NAPIs) for applications in oral hygienic products.

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.

Metabolites profiling of protein enriched oyster mushroom (Pleurotus ostreatus (Jacq.) P. Kumm.) grown on oil palm empty fruit bunch substrate

This study aimed to compare the nutritional composition and metabolites profiles of the Pleurotus ostreatus oyster mushroom grown on oil palm empty fruit bunch (EFB) and rubber wood sawdust (RWS) substrates. The mushrooms were prepared for proximate analysis, beta-glucan content, and metabolites profiling using ultra high performance liquid chromatography-quadrupole time of flights mass spectrometer (UHPLC-QToF). Proximate analysis showed significantly higher crude protein, and beta-glucan contents (p < 0.05) were recovered from the mushrooms grown on EFB substrate as compared to those grown on RWS. Metabolite profiling of hot water extracts showed that three lipids (PE (17:1/0:0), PE (16:1/0:0), and hydroxyoctadecadienoic), two amino acids (acetyl-leucine, and threonic acid), and one polysaccharide (4-O-beta-d-galactopyranosyl-beta-d-xylopyranose) were present at a relatively higher level (maximum fold change >10) in mushrooms grown on EFB. Our results suggest that the metabolites of the mushroom cultivated on EFB substrate were altered. The innovative use of palm oil waste as mushroom cultivation substrate exerts the concept of waste-to-wealth, promoting Sustainable Palm Oil Initiative (SPOI), and enhancing food security through the production of high-protein mushrooms.

N6-Acetyl-L-Lysine and p-Cresol as Key Metabolites in the Pathogenesis of COVID-19 in Obese Patients.

Despite the growing number of the vaccinated population, COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global health burden. Obesity, a metabolic syndrome affecting one-third of the population, has proven to be a major risk factor for COVID-19 severe complications. Several studies have identified metabolic signatures and disrupted metabolic pathways associated with COVID-19, however there are no reports evaluating the role of obesity in the COVID-19 metabolic regulation. In this study we highlight the involvement of obesity metabolically in affecting SARS-CoV-2 infection and the consequent health complications, mainly cardiovascular disease. We measured one hundred and forty-four (144) metabolites using ultra high-performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS) to identify metabolic changes in response to SARS-CoV-2 infection, in lean and obese COVID-19 positive (n=82) and COVID-19 negative (n=24) patients. The identified metabolites are found to be mainly correlating with glucose, energy and steroid metabolisms. Further data analysis indicated twelve (12) significantly yet differentially abundant metabolites associated with viral infection and health complications, in COVID-19 obese patients. Two of the detected metabolites, n6-acetyl-l-lysine and p-cresol, are detected only among the COVID-19 cohort, exhibiting significantly higher levels in COVID-19 obese patients when compared to COVID-19 lean patients. These metabolites have important roles in viral entry and could explain the increased susceptibility of obese patients. On the same note, a set of six metabolites associated with antiviral and anti-inflammatory functions displayed significantly lower abundance in COVID-19 obese patients. In conclusion, this report highlights the plasma metabolome of COVID-19 obese patients as a metabolic feature and signature to help improve clinical outcomes. We propose n6-acetyl-l-lysine and p-cresol as potential metabolic markers which warrant further investigations to better understand their involvement in different metabolic pathways in COVID-19.

The Airway Microbiome and Metabolome in Preterm Infants: Potential Biomarkers of Bronchopulmonary Dysplasia.

ObjectivesWe investigated the genomic and metabolic characteristics of the airway microbiome in mild, moderate, severe, and non-bronchopulmonary dysplasia (BPD) preterm infants and explored possible mechanisms underlying BPD.MethodsTwenty-eight preterm infants with gestational age ≤34 weeks and intubated within 24 h after birth were enrolled. According to the severity of BPD, the patients were divided into mild, moderate and severe BPD groups, and the non-BPD group was the control group. Tracheal aspirates (TA) were obtained at intubation and on day 7 after birth. The bacterium in the aspirates were sequenced by 16S rRNA, and the metabolomics of the aspirates were identified by high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-Q-TOF/MS). The correlation between the differential metabolite and differential bacteria was investigated using Pearson’s correlation coefficient corrected for gestational age and birth weight and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.ResultsThere were significant differences in the diversity and composition of airway microbiome and metabolome between severe, moderate and mild BPD and non-BPD premature infants. At birth (day 1), the difference was more pronounced than at day 7. The diversity of airway microbial community decreased, the abundance of Stenotrophomonas increased, and the increased level of sn-glycerol 3-phosphoethanolamine was positively correlated with the severity of BPD. There was a significant positive correlation between the abundance of Stenotrophomonas and the level of sn-glycerol 3-phosphoethanolamine.ConclusionDecreased diversity of the airway microbiome, increased abundance of Stenotrophomonas, and increased level of sn-glycerol 3-phosphoethanolamine may have potential as biomarkers for BPD. The occurrence and severity of BPD are closely related to Stenotrophomonas, which may influence the composition of the lower airway microbiome through its metabolite sn-glycerol 3-phosphoethanolamine, and may be the triggering factor of the disease. The causal relationship needs further study.

Analysis of the effective components and mechanism of Yufang Fangji II for prevention of COVID-19 based on UHPLC-Q-TOF/MS and network pharmacology

AIM: The main chemical components of Yufang Fangji II (Hubei Fang) of COVID-19 were studied systematically and combined with network pharmacology to provide a reference for the study of its effective substances. METHODS: Ultra high-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-Q-TOF/MS) was applied to identify the absorbed components of the prescription in rat plasma. TCMSP database and Swiss Target Prediction data platform were used to predict the target of the identified blood components, and network visualization software Cytoscape 3.7.2 was used draw the association network diagram, and GO enrichment analysis and KEGG pathway enrichment analysis were conducted for the key targets. With the help of CB-Dock online molecular docking platform, the molecular docking of key targets and blood entering compounds was carried out, and the docking combination with good affinity value was displayed by ligplot software to verify the preventive effect of Yufang Fangji II on COVID-19. RESULTS: A total of 52 chemical components identified in the prescription, in which 13 components were absorbed in the rat plasma as the prototype, and they were from Astragalus membranaceus, Atractylodes macrocephala, Saposhnikoviae Radix, Lonicerae Japonicae Flos, and Citri Reticulatae Pericarpium, respectively. These compounds were recognized to act on 17 core targets, including mapk3, TNF and other targets related to inflammation, MPO and other targets related to oxidative stress, VEGFR, KDR and other targets related to vascular endothelium. The results of molecular docking showed that the absorbed components had good binding activity with the key targets. CONCLUSION: Compounds in Yufang Fangji II are involved in regulating inflammation, oxidative stress, vascular and cellular physiological activities, which have preventive effects on COVID-19 through regulating IL-17, PI3K Akt, MAPK and other pathways.

Structure elucidation of new kinds of bacitracin sulfonic acid based on high performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry

Bacitracin is a mixture of antimicrobial peptides. Although the structures of bacitracins have been fully studied, the structure elucidation of the oxidation products of bacitracin has rarely been reported. In the present work, the bacitracin was treated by hydrogen peroxide solution to form bacitracin sulfonic acids (SO3H) directly, and the structures of bacitracin-SO3Hs were studied by high performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry (HPLC-QTOF-MS/MS). It was demonstrated that the components of bacitracin, such as bacitracin A, B, and C could be site-specifically transformed to the corresponding oxidized product containing the SO3H group. The structure characterization of the oxidized products was elucidated based on the accurate molecular mass and the product ions obtained by MS/MS. Some new kinds of bacitracin J-SO3H, vinyl-bacitracin A-SO3H, bacitracin Y–SO3H and deamino-bacitracin Y–SO3H were identified. Furthermore, we also investigated the novel fragmentation behavior of the oxidized products. This is the first report to propose a neutral loss mechanism of SO3 that explains the characteristic ions of the vinyl-bacitracin A-SO3H and bacitracin Y–SO3H. Based on the structure analysis of the oxidation products of bacitracin, three transformation routes of bacitracin-SO3H generated from bacitracin were hypothesized and systematically summarized for the first time.

Identification of Metabolic Biomarkers in Relation to Methotrexate Response in Early Rheumatoid Arthritis.

This study aimed to identify baseline metabolic biomarkers for response to methotrexate (MTX) therapy in rheumatoid arthritis (RA) using an untargeted method. In total, 82 baseline plasma samples (41 insufficient responders and 41 sufficient responders to MTX) were selected from the Treatment in the Rotterdam Early Arthritis Cohort (tREACH, trial number: ISRCTN26791028) based on patients’ EULAR response at 3 months. Metabolites were assessed using high-performance liquid chromatography-quadrupole time of flight mass spectrometry. Differences in metabolite concentrations between insufficient and sufficient responders were assessed using partial least square regression discriminant analysis (PLS-DA) and Welch’s t-test. The predictive performance of the most significant findings was assessed in a receiver operating characteristic plot with area under the curve (AUC), sensitivity and specificity. Finally, overrepresentation analysis was performed to assess if the best discriminating metabolites were enriched in specific metabolic events. Baseline concentrations of homocystine, taurine, adenosine triphosphate, guanosine diphosphate and uric acid were significantly lower in plasma of insufficient responders versus sufficient responders, while glycolytic intermediates 1,3-/2,3-diphosphoglyceric acid, glycerol-3-phosphate and phosphoenolpyruvate were significantly higher in insufficient responders. Homocystine, glycerol-3-phosphate and 1,3-/2,3-diphosphoglyceric acid were independent predictors and together showed a high AUC of 0.81 (95% CI: 0.72–0.91) for the prediction of insufficient response, with corresponding sensitivity of 0.78 and specificity of 0.76. The Warburg effect, glycolysis and amino acid metabolism were identified as underlying metabolic events playing a role in clinical response to MTX in early RA. New metabolites and potential underlying metabolic events correlating with MTX response in early RA were identified, which warrant validation in external cohorts.

Gliotoxin and bis(methylthio)gliotoxin are not reliable as biomarkers of invasive aspergillosis.

Invasive pulmonary aspergillosis (IPA) remains a life-threatening opportunistic infection, but can be difficult to diagnose. New biomarkers are therefore needed. Gliotoxin (GT), a secondary metabolite of Aspergillus fumigatus, and bis(methylthio)gliotoxin (bmGT), a degradation product of GT, have been proposed as potential biomarkers. However, these findings have yet to be confirmed. To identify the diagnostic potential of GT and bmGT in serum and bronchoalveolar lavage fluid (BALf) in haematology patients compared to galactomannan (GM). We prospectively collected culture supernatant, serum and BALf from patients with culture-positive IPA and measured GT and bmGT concentrations using ultra high-performance liquid chromatography-quadrupole time of flight mass spectrometry. Galactomannan was detected using a commercially available enzyme immunoassay. We included 18 patients with proven (n=6) and probable (n=12) IPA, all with positive cultures for Aspergillus fumigatus. BmGT was only detected in serum from one patient (5.6%), whereas GM was positive (optical density≥0.5) in 11/18 patients (61.1%, P=0.002). We could not find GT in any serum sample. In BALf, bmGT was detected in 8/18 patients (44.4%) and GT in 9/18 patients (50%), compared to GM (optical density≥1.0) in all patients (100%). Gliotoxin and bis(methylthio)gliotoxin had a very poor performance for diagnosing IPA. As other biomarkers are more sensitive and easier to detect, we would not recommend serum or BALf GT/bmGT to be used in the diagnosis of IPA.

Phenolic Metabolites in Plasma and Thigh Meat of Chickens Supplemented with Grape Byproducts.

Grape byproducts are rich sources of polyphenols with powerful antioxidant and health-promoting effects. The impact of supplementing chicken diets with grape byproducts on plasma and thigh meat concentrations of phenolic metabolites was evaluated by analyzing samples by high-performance liquid chromatography quadrupole time of flight mass spectrometry. Chickens were fed three experimental diets: Control diet, Control+8% grape pomace, and Control+0.1% grape seed extract. In plasma, 32 phenolic metabolites were identified, some of which were conjugated catechin/epicatechin metabolites exclusively identified in chickens fed diets enriched in grape byproducts. Also, these chickens showed significantly higher plasmatic concentrations of 21 phenolic metabolites. In thigh meat, 14 phenolic metabolites were identified, but no differences were found between diets. Higher plasmatic tocopherol was found when supplementing diets with grape byproducts, while no changes were observed in meat. Thus, supplementing chicken diets with grape byproducts leads to a significant increase in the circulation of phenolic metabolites and tocopherol.

UHPLC-QTOF/MS-based metabolomics investigation for the protective mechanism of Danshen in Alzheimer's disease cell model induced by Aβ1-42.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder with neither definitive pathogenesis nor effective therapy so far. Danshen, the dried root and rhizome of Salvia miltiorrhiza Bunge, is used extensively in Alzheimer's disease treatment to ameliorate the symptoms, but the underlying mechanism remains to be clarified. To investigate potential biomarkers for AD and elucidate the protective mechanism of Danshen on AD cell model. An ultra high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-QTOF/MS)-based approach combined with partial least squares discriminant analysis (PLS-DA) has been developed to discriminate the metabolic modifications between human brain microvascular endothelial cell (hBMEC) and AD cell model induced by amyloid-β protein (Aβ1-42). To further elucidate the pathophysiology of AD, related metabolic pathways have been studied. Thirty-three distinct potential biomarkers were screened out and considered as potential biomarkers corresponding to AD, which were mostly improved and partially restored back to normalcy in Danshen pre-protection group. It was found that AD was closely related to disturbed arginine and proline metabolism, glutathione metabolism, alanine aspartate and glutamate metabolism, histidine metabolism, pantothenate and CoA biosynthesis, phenylalanine tyrosine and tryptophan biosynthesis, citrate cycle and glycerophospholipid metabolism, and the protective mechanism of Danshen in AD cell model may be related to partially regulating the perturbed pathways. These outcomes provide valuable evidences for therapeutic mechanism investigation of Danshen in AD treatment, and such an approach could be transferred to unravel the mechanism of other traditional Chinese medicine (TCM) and diseases.

Metabolic profiling of Solanum villosum Mill subsp. miniatum (bernh. ex willd.): Hepatoprotective and antifibrotic activity in a rat model of liver fibrosis

Aim/Background: To assess the liver antifibrotic action of ethanolic extract of aerial parts of Solanum villosum Mill subsp. miniatum (Bernh. ex Willd.) (SVE) and correlate this activity with its high performance liquid chromatography-quadrupole time of flight (HPLC-qTOF) Electrospray Ionisation Mass Spectrometry (ESIMS) phytochemical profile. Materials and Methods: The median lethal dose of SVE was determined, and a rat model of carbon tetrachloride (CCl4)-induced liver fibrosis was used to evaluate its antifibrotic activity. Markers for hepatotoxicity, fibrosis, and oxidative stress were assessed, and histopathological features of liver tissues were examined. Metabolite profiling of SVE was achieved via HPLC-qTOF-ESIMS coupled with Photodiode array (PDA). Liver fibrosis was induced in rats by oral administration of CCl4for 6 weeks. Silymarin (positive control) and SVE (100 and 250 mg/kg) were orally administered daily for 6 weeks. Results: Compared to CCl4-intoxicated group, administration of SVE obviously ameliorated fibrosis of the hepatic capsule associated with aggregation of multiple focal fat cells formation. Both silymarin and SVE ameliorated the rise in serum markers of hepatotoxicity (alanine transaminase, aspartate transaminase, and alkaline phosphatase), markedly attenuated CCl4-induced oxidative stress. The antifibrotic activity of SVE was evidenced by inhibiting the rise in hepatic hydroxyproline content and accumulation of collagen. This was confirmed by the ability of SVE to inhibit alterations in expression of the fibrosis-related genes Collagen Iα, matrixmetalloproteinase-2 (MMP-2), tissue inhibitor matrix metalloproteinase-2, and transforming growth factor beta 1. HPLC-qTOF-ESIMS analysis of SVE revealed the presence of 47 metabolites, among which 33 were tentatively identified. Conclusion: SVE exhibited hepatoprotective and antifibrotic activities in rats by enhancing the antioxidant capacity and regulating expression of fibrogenic mediators.

Shufeng Jiedu Capsules Alleviate Lipopolysaccharide-Induced Acute Lung Inflammatory Injury via Activation of GPR18 by Verbenalin

Background/Aims: Acute respiratory tract infection (ARTI) is the most common reason for outpatient physician office visits. Although powerful and significant in the treatment of infections, antibiotics used for ARTI inappropriately have been an important contributor to antibiotic resistance. We previously reported that Shufeng Jiedu Capsule (SJC) can effectively amplify anti-inflammatory signaling during infection. In this study, we aimed to systematically explore its composition and the mechanism of its effects in ARTI. Methods: Pseudomonas aeruginosa (PAK) strain was used to generate a mouse model of ARTI, which were then treated with different drugs or compounds to determine the corresponding anti-inflammatory roles. High-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry. was conducted to detect the chemical compounds in SJC. RNAs from the lung tissues of mice were prepared for microarray analysis to reveal globally altered genes and the pathways involved after SJC treatment. Results: SJC significantly inhibited the expression and secretion of inflammatory factors from PAK-induced mouse lung tissues or lipopolysaccharide-induced peritoneal macrophages. Verbenalin, one of the bioactive compounds identified in SJC, also showed notable anti-inflammatory effects. Microarray data revealed numerous differentially expressed genes among the different treatment groups; here, we focused on studying the role of GPR18. We found that the anti-inflammatory role of verbenalin was attenuated in GPR18 knockout mice compared with wild-type mice, although no statistically significant difference was observed in the untreated PAK-induced mice types. Conclusion: Our data not only showed the chemical composition of SJC, but also demonstrated that verbenalin was a significant anti-inflammatory compound, which may function through GPR18.

Comprehensive quantitative analysis of Chinese patent drug YinHuang drop pill by ultra high-performance liquid chromatography quadrupole time of flight mass spectrometry

YinHuang drop pill (YHDP) is a new preparation, derived from the traditional YinHuang (YH) decoction. Since drop pills are one of the newly developed forms of Chinese patent drugs, not much research has been done regarding the quality and efficacy. This study aims to establish a comprehensive quantitative analysis of the chemical profile of YHDP. ultra high-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS/MS) was used to identify 34 non-sugar small molecules including 15 flavonoids, 9 phenolic acids, 5 saponins, 1 iridoid, and 4 iridoid glycosides in YHDP samples, and 26 of them were quantitatively determined. Sugar composition of YHDP in terms of fructose, glucose and sucrose was examined via a high performance liquid chromatography-evaporative light scattering detector on an amide column (HPLC-NH2P-ELSD). Macromolecules were examined by high performance gel permeation chromatography coupled with ELSD (HPGPC-ELSD). The content of the drop pill’s skeleton component PEG-4000 was also quantified via ultra-high performance liquid chromatography coupled with charged aerosol detector (UHPLC-CAD). The results showed that up to 73% (w/w) of YHDP could be quantitatively determined. Small molecules accounted for approximately 5%, PEG-4000 represented 68%, while no sugars or macromolecules were found. Furthermore, YHDP showed no significant differences in terms of daily dosage, compared to YinHuang granules and YinHuang oral liquid; however, it has a higher small molecules content compared to YinHuang lozenge.

Traditional Aboriginal Preparation Alters the Chemical Profile of Carica papaya Leaves and Impacts on Cytotoxicity towards Human Squamous Cell Carcinoma

Carica papaya leaf decoction, an Australian Aboriginal remedy, has been used widely for its healing capabilities against cancer, with numerous anecdotal reports. In this study we investigated its in vitro cytotoxicity on human squamous cell carcinoma cells followed by metabolomic profiling of Carica papaya leaf decoction and leaf juice/brewed leaf juice to determine the effects imparted by the long heating process typical of the Aboriginal remedy preparation. MTT assay results showed that in comparison with the decoction, the leaf juice not only exhibited a stronger cytotoxic effect on SCC25 cancer cells, but also produced a significant cancer-selective effect as shown by tests on non-cancerous human keratinocyte HaCaT cells. Furthermore, evidence from testing brewed leaf juice on these two cell lines suggested that the brewing process markedly reduced the selective effect of Carica papaya leaf on SCC25 cancer cells. To tentatively identify the compounds that contribute to the distinct selective anticancer activity of leaf juice, an untargeted metabolomic approach employing Ultra High Performance Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry followed by multivariate data analysis was applied. Some 90 and 104 peaks in positive and negative mode respectively were selected as discriminatory features from the chemical profile of leaf juice and >1500 putative compound IDs were obtained via database searching. Direct comparison of chromatographic and tandem mass spectral data to available reference compounds confirmed one feature as a match with its proposed authentic standard, namely pheophorbide A. However, despite pheophorbide A exhibiting cytotoxic activity on SCC25 cancer cells, it did not prove to be the compound contributing principally to the selective activity of leaf juice. With promising results suggesting stronger and more selective anticancer effects when compared to the Aboriginal remedy, Carica papaya leaf juice warrants further study to explore its activity on other cancer cell lines, as well as investigation to confirm the identity of compounds contributing to its selective effect, particularly those compounds altered by the long heating process applied during the traditional Aboriginal remedy preparation.

Chemical Characterization and in Vitro Cytotoxicity on Squamous Cell Carcinoma Cells of Carica Papaya Leaf Extracts

In traditional medicine, Carica papaya leaf has been used for a wide range of therapeutic applications including skin diseases and cancer. In this study, we investigated the in vitro cytotoxicity of aqueous and ethanolic extracts of Carica papaya leaves on the human oral squamous cell carcinoma SCC25 cell line in parallel with non-cancerous human keratinocyte HaCaT cells. Two out of four extracts showed a significantly selective effect towards the cancer cells and were found to contain high levels of phenolic and flavonoid compounds. The chromatographic and mass spectrometric profiles of the extracts obtained with Ultra High Performance Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry were used to tentatively identify the bioactive compounds using comparative analysis. The principal compounds identified were flavonoids or flavonoid glycosides, particularly compounds from the kaempferol and quercetin families, of which several have previously been reported to possess anticancer activities. These results confirm that papaya leaf is a potential source of anticancer compounds and warrant further scientific investigation to validate the traditional use of papaya leaf to treat cancer.

Accurate Mass GC/LC-Quadrupole Time of Flight Mass Spectrometry Analysis of Fatty Acids and Triacylglycerols of Spicy Fruits from the Apiaceae Family.

The triacylglycerol (TAG) structure and the regio-stereospecific distribution of fatty acids (FA) of seed oils from most of the Apiaceae family are not well documented. The TAG structure ultimately determines the final physical properties of the oils and the position of FAs in the TAG molecule affects the digestion; absorption and metabolism; and physical and technological properties of TAGs. Fixed oils from the fruits of dill (Anethum graveolens), caraway (Carum carvi), cumin (Cuminum cyminum), coriander (Coriandrum sativum), anise (Pimpinella anisum), carrot (Daucus carota), celery (Apium graveolens), fennel (Foeniculum vulgare), and Khella (Ammi visnaga), all from the Apiaceae family, were extracted at room temperature in chloroform/methanol (2:1 v/v) using percolators. Crude lipids were fractionated by solid phase extraction to separate neutral triacylglycerols (TAGs) from other lipids components. Neutral TAGs were subjected to transesterification process to convert them to their corresponding fatty acids methyl esters (FAMES) using 1% boron trifluoride (BF3) in methanol. FAMES were analyzed by gas chromatography-quadrupole time of flight (GC-QTOF) mass spectrometry. Triglycerides were analyzed using high performance liquid chromatography-quadrupole time of flight (LC-QTOF) mass spectrometry. Petroselinic acid was the major fatty acid in all samples ranging from 57% of the total fatty acids in caraway up to 82% in fennel. All samples contained palmitic (16:0), palmitoleic (C16:1n-9), stearic (C18:0), petroselinic (C18:1n-12), linoleic (C18:2n-6), linolinic (18:3n-3), and arachidic (C20:0) acids. TAG were analyzed using LC-QTOF for accurate mass identification and mass spectrometry/mass spectrometry (MS/MS) techniques for regiospesific elucidation of the identified TAGs. Five major TAGs were detected in all samples but with different relative concentrations in all of the tested samples. Several other TAGs were detected as minor components and were present in some samples and absence in the others. Regiospecific analysis showed a non-random fatty acids distribution. Petroselinic acid was predominantly located at the sn-1 and sn-3 positions.

Metabolite profiling and pharmacokinetic evaluation of hydrocortisone in a perfused three-dimensional human liver bioreactor.

Endotoxin lipopolysaccharide (LPS) is known to cause liver injury primarily involving inflammatory cells such as Kupffer cells, but few in vitro culture models are applicable for investigation of inflammatory effects on drug metabolism. We have developed a three-dimensional human microphysiological hepatocyte-Kupffer cell coculture system and evaluated the anti-inflammatory effect of glucocorticoids on liver cultures. LPS was introduced to the cultures to elicit an inflammatory response and was assessed by the release of proinflammatory cytokines, interleukin 6 and tumor necrosis factor α. A sensitive and specific reversed-phase-ultra high-performance liquid chromatography-quadrupole time of flight-mass spectrometry method was used to evaluate hydrocortisone disappearance and metabolism at near physiologic levels. For this, the systems were dosed with 100 nM hydrocortisone and circulated for 2 days; hydrocortisone was depleted to approximately 30 nM, with first-order kinetics. Phase I metabolites, including tetrahydrocortisone and dihydrocortisol, accounted for 8-10% of the loss, and 45-52% consisted of phase II metabolites, including glucuronides of tetrahydrocortisol and tetrahydrocortisone. Pharmacokinetic parameters, i.e., half-life, rate of elimination, clearance, and area under the curve, were 23.03 hours, 0.03 hour(-1), 6.6 × 10(-5) l⋅hour(-1), and 1.03 (mg/l)*h, respectively. The ability of the bioreactor to predict the in vivo clearance of hydrocortisone was characterized, and the obtained intrinsic clearance values correlated with human data. This system offers a physiologically relevant tool for investigating hepatic function in an inflamed liver.

Rapid characterization of impurities in the bulk drug of nifedipine by high performance liquid chromatography-quadrupole time of fight mass spectrometry

Four impurities in the bulk drug of nifedipine were detected by means of high performance liquid chromatography coupled with quadrupole time of fight mass spectrometry (HPLC-QTOF MS). A Kromasil C18 HPLC column (250 mm x 4.6 mm, 5 microm) was used with methanol-water (60:40, v/v) as mobile phase. The column temperature was maintained at 35 degrees C with an ultraviolet (UV) detection wavelength of 235 nm for analysis. The eluates were detected with a UV detector and a quadrupole time of fight mass spectrometer in positive mode. The sample was infused into the mass spectrometer from the HPLC system through a T-junction with a splitting ratio of 3:1. The ion source temperature was set at 320 degrees C and the electrospray ionization (ESI) needle voltage was always set at 4 000 V. Nitrogen was used as the drying gas and nebulizer gas. The protonated molecular ions were selected for determining the structures, and their fragmentation pathways were deduced. Based on the fragmentation behavior of nifedipine, the structures of 3 impurities were identified and one of them was identified as 3-ethyl-5-methyl-2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate, which was an unknown impurity to the best of our knowledge. An other impurity was identified by the comparison of the retention time and tandem mass spectra with the standard substance. The four proposed structures were all confirmed by exact mass evidence. The results indicated that the described method can be effectively applied to perform the identifications in nifedipine and its impurities.

Identification of substituted cathinones: 3,4-Methylenedioxy derivatives by high performance liquid chromatography–quadrupole time of flight mass spectrometry

The potential of high performance liquid chromatography–high resolution mass spectrometry (HPLC–HRMS) for screening of synthetic cathinones in legal highs was examined. Samples were analysed by liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometer (Q/TOF). Nanoelectrospray ionisation (nanoESI) was employed. MS and MS/MS spectra were acquired. Six 3,4-methylenedioxy derivatives: methylone, butylone, pentylone, MDPBP, MDPV and BMDP were detected and identified. The fragmentation pattern of 3,4-methylenedioxy derivatives in collision induced dissociation (CID) was derived and described, which will facilitate future screenings and identifications of new synthetic cathinones. For 3,4-methylenodioxy derivative cathinones the loss of neutral groups CH4O2, H2O, amines and imines is observed. The loss of water and the methylenedioxy group does not occur when cyclic amino group – pyrrolidynyl is present in the molecule. Phenyloxazole cations are formed when CH4O2 is lost. The formation of the metylenedioxybenzoyloxonium and allyldioxybenzoyloxonium ions is typical for 3,4-methylenodioxy derivatives, however, the formation of the former appears to be inhibited by the presence in the molecule of the group of atoms able to form very stable tropylium carbocation.