Untargeted Metabolomic Analysis of Leaves and Roots of Jatropha curcas Genotypes with Contrasting Levels of Phorbol Esters.

AIMS: Phorbol esters (PE) are toxic diterpenoids accumulated in physic nut (Jatropha curcas L.) seed tissues. Their biosynthetic pathway remains unknown, and the participation of roots in this process may be possible. Thus, we set out to study the deposition pattern of PE and other terpenoids in roots and leaves of genotypes with detected (DPE) and not detected (NPE) phorbol esters based on previous studies. OUTLINE OF DATA RESOURCES: We analyzed physic nut leaf and root organic extracts using LC-HRMS. By an untargeted metabolomics approach, it was possible to annotate 496 and 146 metabolites in the positive and negative electrospray ionization modes, respectively. KEY RESULTS: PE were detected only in samples of the DPE genotype. Remarkably, PE were found in both leaves and roots, making this study the first report of PE in J. curcas roots. Furthermore, untargeted metabolomic analysis revealed that diterpenoids and apocarotenoids are preferentially accumulated in the DPE genotype in comparison with NPE, which may be linked to the divergence between the genotypes concerning PE biosynthesis, since sesquiterpenoids showed greater abundance in the NPE. UTILITY OF THE RESOURCE: The LC-HRMS files, publicly available in the MassIVE database (identifier MSV000092920), are valuable as they expand our understanding of PE biosynthesis, which can assist in the development of molecular strategies to reduce PE levels in toxic genotypes, making possible the food use of the seedcake, as well as its potential to contain high-quality spectral information about several other metabolites that may possess biological activity.

Statine-based peptidomimetic compounds as inhibitors for SARS-CoV-2 main protease (SARS-CoV­2 Mpro).

COVID-19 is a multisystemic disease caused by the SARS-CoV-2 airborne virus, a member of the Coronaviridae family. It has a positive sense single-stranded RNA genome and encodes two non-structural proteins through viral cysteine-proteases processing. Blocking this step is crucial to control virus replication. In this work, we reported the synthesis of 23 statine-based peptidomimetics to determine their ability to inhibit the main protease (Mpro) activity of SARS-CoV-2. Among the 23 peptidomimetics, 15 compounds effectively inhibited Mpro activity by 50% or more, while three compounds (7d, 8e, and 9g) exhibited maximum inhibition above 70% and IC50 < 1 µM. Compounds 7d, 8e, and 9g inhibited roughly 80% of SARS-CoV-2 replication and proved no cytotoxicity. Molecular docking simulations show putative hydrogen bond and hydrophobic interactions between specific amino acids and these inhibitors. Molecular dynamics simulations further confirmed the stability and persisting interactions in Mpro's subsites, exhibiting favorable free energy binding (ΔGbind) values. These findings suggest the statine-based peptidomimetics as potential therapeutic agents against SARS-CoV-2 by targeting Mpro.

Critical Factors in Sample Collection and Preparation for Clinical Metabolomics of Underexplored Biological Specimens.

This review article compiles critical pre-analytical factors for sample collection and extraction of eight uncommon or underexplored biological specimens (human breast milk, ocular fluids, sebum, seminal plasma, sweat, hair, saliva, and cerebrospinal fluid) under the perspective of clinical metabolomics. These samples are interesting for metabolomics studies as they reflect the status of living organisms and can be applied for diagnostic purposes and biomarker discovery. Pre-collection and collection procedures are critical, requiring protocols to be standardized to avoid contamination and bias. Such procedures must consider cleaning the collection area, sample stimulation, diet, and food and drug intake, among other factors that impact the lack of homogeneity of the sample group. Precipitation of proteins and removal of salts and cell debris are the most used sample preparation procedures. This review intends to provide a global view of the practical aspects that most impact results, serving as a starting point for the designing of metabolomic experiments.

High-speed countercurrent chromatography with offline detection by electrospray mass spectrometry and nuclear magnetic resonance detection as a tool to resolve complex mixtures: A practical approach using Coffea arabica leaf extract.

INTRODUCTION: Many secondary metabolites isolated from plants have been described in the literature owing to their important biological properties and possible pharmacological applications. However, the identification of compounds present in complex plant extracts has remained a great scientific challenge, is often laborious, and requires a long research time with high financial cost. OBJECTIVES: The aim of this study was to develop a method that allows the identification of secondary metabolites in plant extracts with a high degree of confidence in a short period of time. MATERIAL AND METHODS: In this study, an ethanolic extract of Coffea arabica leaves was used to validate the proposed method. Countercurrent chromatography was chosen as the initial step for extraction fractionation using gradient elution. Resulting fractions presented a variation of compounds concentrations, allowing for statistical total correlation spectroscopy (STOCSY) calculations between liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) and NMR across fractions. RESULTS: The proposed method allowed the identification of 57 compounds. Of the annotated compounds, 20 were previously described in the literature for the species and 37 were reported for the first time. Among the inedited compounds, we identified flavonoids, alkaloids, phenolic acids, coumarins, and terpenes. CONCLUSION: The proposed method presents itself as a valid alternative for the study of complex extracts in an effective, fast, and reliable way that can be reproduced in the study of other extracts.

Development of a kit for urine collection on filter paper as an alternative for Pompe disease screening and monitoring by LC-HRMS.

Pompe disease (PD) is an inborn error of metabolism caused by α-glucosidase acid enzyme deficiency. It significantly impacts patients' health and life quality and may lead to death in the first few years of life. Among the well-established diagnostic methods, urinary glucose tetrasaccharide (Glc4) screening by high performance-liquid chromatography has been helpful in monitoring Glc4 levels in patients on enzyme replacement therapy, demonstrating therapy efficacy. However, the specimen shipping process from a sample collecting location to a specialized laboratory for monitoring the Glc4 is costly and presents preanalytical challenges. In this work, we developed a filter paper based-urine collection kit to facilitate specimen shipment, and liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) analysis to determine Glc4 and creatinine in dried urine on filter paper. The LC-HRMS was based on a combination of targeted and untargeted screening on the same specimen injection and was successfully developed and validated. Bland-Altman statistics revealed a good relationship between dried and liquid urine samples and Glc4 and creatinine. Glc4 and other metabolites in dried urine showed stability for at least 7 days at 4 and 22 °C, and 3 days at 50 °C. The stability of the analytes and the efficiency of the kit were tested simulating real conditions by sending it by post. After two days in transit without refrigeration, the stability of compounds was maintained, showing the reliability of the urine collection kit and analysis method to determine the PD biomarker Glc4.

Integrated NMR and MS Analysis of the Plasma Metabolome Reveals Major Changes in One-Carbon, Lipid, and Amino Acid Metabolism in Severe and Fatal Cases of COVID-19.

Brazil has the second-highest COVID-19 death rate worldwide, and Rio de Janeiro is among the states with the highest rate in the country. Although vaccine coverage has been achieved, it is anticipated that COVID-19 will transition into an endemic disease. It is concerning that the molecular mechanisms underlying clinical evolution from mild to severe disease, as well as the mechanisms leading to long COVID-19, are not yet fully understood. NMR and MS-based metabolomics were used to identify metabolites associated with COVID-19 pathophysiology and disease outcome. Severe COVID-19 cases (n = 35) were enrolled in two reference centers in Rio de Janeiro within 72 h of ICU admission, alongside 12 non-infected control subjects. COVID-19 patients were grouped into survivors (n = 18) and non-survivors (n = 17). Choline-related metabolites, serine, glycine, and betaine, were reduced in severe COVID-19, indicating dysregulation in methyl donors. Non-survivors had higher levels of creatine/creatinine, 4-hydroxyproline, gluconic acid, and N-acetylserine, indicating liver and kidney dysfunction. Several changes were greater in women; thus, patients' sex should be considered in pandemic surveillance to achieve better disease stratification and improve outcomes. These metabolic alterations may be useful to monitor organ (dys) function and to understand the pathophysiology of acute and possibly post-acute COVID-19 syndromes.

A Scoping Review of Genus Viscum: Biological and Chemical Aspects of Alcoholic Extracts.

The genus Viscum comprises a large number of semi-parasitic shrubs popularly known as Mistletoe. The Viscum species grow in many countries of Europe, Africa and Asia with different popular uses in ornamentation, foods and medicine. Many studies about Viscum have been done over the last years focusing on biological activities and chemical composition of the aqueous extracts, mainly related to anthroposophical medicines. However, it is known that non-aqueous preparations, as alcoholic extracts, have demonstrated different biological activities that are species-and host tree-dependent. Considering the potential of these alcoholic extracts, a scoping review was conducted using data from three online databases: PubMed, Scopus and Embase. Inclusion criteria consisted of the in vitro, in vivo, ex vivo, clinical and chemical studies of alcoholic extracts from Viscum species. The present review summarized 124 original publications about fourteen Viscum species. Viscum album, Viscum articulatum and Viscum coloratum were the main studied species. Alcoholic extracts demonstrated hypotensive, anticancer, antimicrobial, analgesic and anti-inflammatory capabilities, among other biological activities. Flavonoids, phenolic acids and terpenoids represented 48%, 24% and 11% of the total identified compounds, respectively. This review contributes to the knowledge of alcoholic preparations of the Viscum species and points out the lack of clinical studies concerning these different extracts.

Combined targeted and untargeted high-resolution mass spectrometry analyses to investigate metabolic alterations in pompe disease.

INTRODUCTION: Pompe disease is a rare, lysosomal disorder, characterized by intra-lysosomal glycogen accumulation due to an impaired function of α-glucosidase enzyme. The laboratory testing for Pompe is usually performed by enzyme activity, genetic test, or urine glucose tetrasaccharide (Glc4) screening by HPLC. Despite being a good preliminary marker, the Glc4 is not specific for Pompe. OBJECTIVE: The purpose of the present study was to develop a simple methodology using liquid chromatography-high resolution mass spectrometry (LC-HRMS) for targeted quantitative analysis of Glc4 combined with untargeted metabolic profiling in a single analytical run to search for complementary biomarkers in Pompe disease. METHODS: We collected 21 urine specimens from 13 Pompe disease patients and compared their metabolic signatures with 21 control specimens. RESULTS: Multivariate statistical analyses on the untargeted profiling data revealed Glc4, creatine, sorbitol/mannitol, L-phenylalanine, N-acetyl-4-aminobutanal, N-acetyl-L-aspartic acid, and 2-aminobenzoic acid as significantly altered in Pompe disease. This panel of metabolites increased sample class prediction (Pompe disease versus control) compared with a single biomarker. CONCLUSION: This study has demonstrated the potential of combined acquisition methods in LC-HRMS for Pompe disease investigation, allowing for routine determination of an established biomarker and discovery of complementary candidate biomarkers that may increase diagnostic accuracy, or improve the risk stratification of patients with disparate clinical phenotypes.

Untargeted LC-HRMS metabolomics reveals candidate biomarkers for mucopolysaccharidoses.

BACKGROUND: Mucopolysaccharidoses (MPSs) are inherited genetic diseases caused by an absence or deficiency of lysosomal enzymes responsible for catabolizing glycosaminoglycans (GAGs). Undiagnosed patients, or those without adequate treatment in early life, can be severely and irreversibly affected by the disease. In this study, we applied liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics to identify potential biomarkers for MPS disorders to better understand how MPS may affect the metabolome of patients. METHODS: Urine samples from 37 MPS patients (types I, II, III, IV, and VI; untreated and treated with enzyme replacement therapy (ERT)) and 38 controls were analyzed by LC-HRMS. Data were processed by an untargeted metabolomics workflow and submitted to multivariate statistical analyses to reveal significant differences between the MPS and control groups. RESULTS: A total of 12 increased metabolites common to all MPS types were identified. Dipeptides, amino acids and derivatives were increased in the MPS group compared to controls. N-acetylgalactosamines 4- or 6-sulfate, important constituents of GAGs, were also elevated in MPS patients, most prominently in those with MPS VI. Notably, treated patients exhibited lower levels of the aforementioned acylaminosugars than untreated patients in all MPS types. CONCLUSIONS: Untargeted metabolomics has enabled the detection of metabolites that could improve our understanding of MPS physiopathology. These potential biomarkers can be utilized in screening methods to support diagnosis and ERT monitoring.

Neotropical mustelids: fecal metabolome diversity and its potential for taxonomic discrimination.

Chemical profiles of non-invasive biological material, such as feces, have great potential to study elusive animals or those with low population densities. Here, we use a metabolomic approach to evaluate Neotropical mustelids as a biological model to describe the diversity of the metabolites present in fecal samples, as well as to evaluate the potential of chemical profiles for taxonomic discrimination. We collected fecal samples from captive individuals of 5 species of mustelids occurring in Brazil and analyzed them by liquid chromatography coupled to high-resolution mass spectrometry. Over 200 compounds have been annotated; "bile acids, alcohols and derivatives" was the most expressive class in the metabolome of all the species. We successfully discriminated 3 taxonomic groups: 1-tayra (Eira barbara); 2-otters (Lontra longicaudis and Pteronura brasiliensis; 1); and 3-grisons (Galictis vittata and Galictis cuja). Several compounds seemed to be associated with food intake and the digestive process, while others were found for the first time in Neotropical mustelids. We concluded that mustelids show high metabolome diversity and that species-specific identification through metabolomic profiles is possible, thus contributing to the development and implementation of additional non-invasive approaches in the study of mustelids.

Lecithin-based nanocapsule loading sucupira (Pterodon emarginatus) oil effects in experimental mucositis.

Intestinal mucositis (IM) is a frequent adverse effect in anticancer therapy without standard treatment. The oil obtained from sucupira (Pterodon emarginatus) has anti-inflammatory properties, and the soybean lecithin reduces the intestinal toxicity of several xenobiotics. However, their water insolubility impairs the in vivo application. For this reason, we evaluated if the nanoencapsulation of sucupira oil (SO) in lecithin-based nanocapsules (SO-NC) could be a therapeutically effective system for the treatment of IM in murine cisplatin (CDDP)-induced intestinal mucositis model. SO was analyzed by LC-HRMS/MS and HPLC. SO-NC was prepared by nanoprecipitation and characterized using DLS, HPLC, and AFM. Mice body weight and food consumption were assessed daily during experimental mucositis induced by CDDP. The animals were euthanized, and intestinal permeability, inflammatory mediators, and intestinal histology were performed. SO-NC demonstrated adequate characteristics for oral administration as size under 300 nm, IP < 0.3, high EE, and spherical shape. In vitro cytotoxicity performed against RAW 264.7 cell lines resulted in cell viability above 80 % confirming the non-cytotoxic profile of SO (IC50 268 µg/mL) and SO-NC (IC50 118.5 µg/mL) up to 117.2 µg/mL. The untreated mice showed intestinal toxicity after i.p. of CDDP, principally weight loss, increased intestinal permeability, and MPO and TNF-α levels. Surprisingly, the administration of SO to CDDP-mucositis animals did not circumvent the CDDP effects and increased intestinal permeability. However, SO-NC proved efficient in mitigating the experimental intestinal mucositis by improving intestinal epithelium architecture, reducing intestinal permeability, and improving the MPO levels. In conclusion, SO-NC can positively impact intestinal mucositis by promoting mucosal recovery. This is a promising strategy for developing a new treatment for intestinal mucositis.

Viscum album mother tinctures: Harvest conditions and host trees influence the plant metabolome and the glycolytic pathway of breast cancer cells.

Viscum album is a semi-parasitic plant used for over one hundred years in complementary cancer therapy. The main commercial drugs used in cancer patients' treatment are derived from the aqueous V. album extracts, whose cytotoxic potential is mostly attributed to the aqueous soluble antitumoral metabolites. On the counterpart, ethanol solvents must be used to obtain V. album mother tinctures. This methodology permits better solubilization of phenolic compounds, among others, which present antitumoral bioactivity. Recently, the metabolomics approach revealed the influence of the host tree on the V. album subspecies differentiation. To increase the scientific information about the chemical differences related to the host trees and to clarify the seasonal influences, in this study, the metabolome of 50 V. album mother tinctures from three subspecies (abietis, album, austriacum) and five host trees (Malus domestica, Quercus sp., Ulmus carpinifolia, Pinus sylvestris, Abies alba) was evaluated using summer and winter plant harvests. The in vitro cytotoxic activities were investigated in breast cancer cells (MDA-MB-231) and immortalized normal human keratinocytes (HaCaT). The summer V. album mother tinctures presented higher cytotoxic activity than winter ones. Among the summer samples, those prepared with V. album subsp. album were more cytotoxic than V. album subsp. abietis and subsp. V. album subsp. austriacum. The V. album harvested from Quercus petraea and Abies alba inhibited the key-glycolytic enzymes: hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK). This activity was related to a reduction in glucose uptake and lactate production, which were host-tree-time-dose-dependent. The untargeted metabolomic approach was able to discriminate the mother tinctures according to respective botanical classes and harvest season. A total of 188 metabolites were annotated under positive and negative modes. Fourteen compounds were responsible for the samples differentiation, and, to the best of our knowledge, eight were described in the Viscum album species for the first time. Our study shows the interruption of the Warburg effect as a novel antitumoral mechanism triggered by V. album mother tinctures, which is related to their metabolite profile. These results bring scientific evidence that encourages the use of V. album mother tinctures as a natural product for cancer therapy.

Metabology: Analysis of metabolomics data using community ecology tools.

Several areas such as microbiology, botany, and medicine use genetic information and computational tools to organize, classify and analyze data. However, only recently has it been possible to obtain the chemical ontology of metabolites computationally. The systematic classification of metabolites into classes opens the way for adapting methods that previously used genetic taxonomy to now accept chemical ontology. Community ecology tools are ideal for this adaptation as they have mature methods and enable exploratory data analysis with established statistical tools. This study introduces the Metabology approach, which transforms metabolites into an ecosystem where the metabolites (species) are related by chemical ontology. In the present work, we demonstrate the applicability of this new approach using publicly available data from a metabolomics study of human plasma that searched for prognostic markers of COVID-19, and in an untargeted metabolomics study carried out by our laboratory using Lasiodiplodia theobromae fungal pathogen supernatants.

Combining high-speed countercurrent chromatography three-phase solvent system with electrospray ionization-mass spectrometry and nuclear magnetic resonance to profile the unconventional food plant Syzygium malaccense.

Syzygium malaccense (L.) Merr. & L.M. Perry is a native tree to Malaysia, but also occurs in other tropical regions of the world, including Brazil. The increasing interest in the consumption of its leaves motivated the investigation of compounds of the plant. Metabolite profiling of S. malaccense leaves was achieved by high-speed countercurrent chromatography (HSCCC) fractionation coupled off-line to electrospray mass-spectrometry (ESI-MS) detection and nuclear magnetic resonance (NMR) analysis. The ethanolic leaf extract was submitted to HSCCC using a three-phase solvent system (TPSS) composed by n-hexane - ethyl acetate - acetonitrile - H2O (2:1:1:1, v/v). The stepwise gradient elution was employed due to the extract's chemical complexity. HSCCC fractions were further analyzed by ESI-MS/MS using a flow injection experiment and by NMR acquiring 1H, HSQC and HMBC spectra. MS based dereplication was achieved by comparing acquired data to those available in public and commercial databases. Results were also correlated to previously isolated compounds described for the Syzygium genus. This process led to the annotation of 90 compounds. The NMR data provided structural confirmation and substitution patterns for some of them. Extract chemical composition is characterized by having flavonoids, benzoic acids, hydroxycinnamic acids, quinic acids, hydrolizable tannins, fatty acids, anacardic acids and others primary metabolites. Most of these compounds were described for the first time in the plant. This approach greatly facilitates phytochemical analysis and could be applied to improve metabolite discovery in other studies.

Viscumalbum L. homeopathic mother tinctures: metabolome and antitumor activity

Viscum album L. is a semi-parasitic plant with antitumor activity attributed to theaqueous extracts. However, European V. album ethanolic extracts (VAE) have also demonstrated invitro activity in tumor models. Aims: Evaluate the metabolic profiles of fifty VAE harvested duringsummer and winter seasons and their antitumor activity through 2D and 3D models. Methodology:VAEwerepreparedbymacerationfrom:V.albumsubsp.albumgrowingonMalus domestica,Quercus sp.and Ulmus sp.; V. album subsp. austriacum from Pinus sylvestris; V. album subsp. abietis from Abies alba.Chemical analyses were performed through liquid chromatography coupled with high resolutionmass spectrometry and Partial Least Squares Discriminant Analysis (PLS-DA) was performed in theMetaboanalyst 4.0. The antitumor potential of the selected VAE was evaluated in 2D and 3D models(MDA-MB-231 cancer cells) by MTT, crystal violet and glycolytic pathway analysis. Results anddiscussion:Thefirst3principalcomponentsinPLS-DAexplained60%and40%ofdatavariationin positive andnegativemodesrespectively.Threegroupswereformedandshowedchemicalsimilarityamong V. album subspecies. The compounds responsible for group separation were tentativelyidentifiedas:pinobankasinornaringenin hexoside;isorhamnetin-3-hexoside,meglutolanddifferent aminoacids.ThesummerVAEat0.5%v/vinducedhighercytotoxicdamagethanthewinterpreparations, and Abies alba and Quercus sp. VAE promoted 49% and 42% reduction of tumorviability in 3D model (72h incubation), respectively. MDA-MB-231 glycolytic pathway in 2D modelshowed a decrease in the glucose consumption and extracellular lactate production. Also, PFK (6-phosphofructo-1-kinase)andPK(Pyruvatekinase)activitieswereinhibitedbyAbiesalbaandQuercus sp. VAE at 48h of incubation. Conclusion: VAE extracts showed different metabolomes andthe glycolytic pathway should be an important target involved in the inhibition of tumor growth bytheseextracts

Vanilla flavor: Species from the Atlantic forest as natural alternatives.

The volatility of the vanilla market calls attention to the so-called vanilla crisis. There is a growing worldwide demand for natural vanilla with a concomitant reduction in global supply. However, commercial crops are threatened with extinction due to the lack of gene pool variability, susceptibility to climate change and pandemic diseases. Therefore, there is an urgent need to identify new Vanilla spp. as alternative sources vanilla. Therefore, using undirected LC-MS/MS metabolic profiling and chemometrics, the present study demonstrates the great bioeconomic potential of two Atlantic Forest species - V. bahiana and V. chamissonis - by annotation of important flavor compounds associated with the commercial species and reveals distinct flavor descriptors associated with both wild species. Such similarities and dissimilarities are crucial to the ongoing quest to Vanilla gene pool improvement. Compounds remarkably and frequently associated with vanilla flavor were annotated or identified in this study such as vanillin and p-hydroxybenzaldehyde.

Metabolomics by UHPLC-Q-TOF Reveals Host Tree-Dependent Phytochemical Variation in Viscum album L.

Viscum album L., commonly known as European mistletoe, is a hemi-parasitic plant of the Santalaceae family. The in vitro and in vivo effects of V. album differ, according to its host tree. However, little is known about the host-dependent phytochemical diversity in V. album. In this study, the metabolic profiles of V. album ssp. album from Malus domestica Bork., Quercus robur L., and Ulmus carpinifolia Gled were compared. Leaves, stems, and berries were collected in Switzerland, by the same procedure, in September 2016 and 2017. The methanolic extracts were analyzed by ultra-performance liquid chromatography, coupled to electrospray quadrupole time-of-flight mass spectrometry in positive ionization mode. The data were submitted to partial-least square discriminant analysis (PLS-DA) and the results showed that the V. album ssp. album samples were clustered into three groups, according to the three distinct host trees. Seven compounds, with high VIP scores (variable importance in projection), were responsible for this differentiation. The following four compounds were detected in both the harvest years: arginine, pipecolic acid or lysine, dimethoxycoumarin, and sinapyl alcohol, suggesting their use as host specific V. album biomarkers. The present work highlights the importance of standardized harvest and analytical procedures for the reproducibility of the chemical results of herbal materials.

Pomegranate (Punica granatum) peel fractions obtained by supercritical CO2 increase oxidative and colour stability of bluefish (Pomatomus saltatrix) patties treated by UV-C irradiation.

The sequential fractionation by supercritical-CO2 (SC-CO2) was applied to obtain fractions enriched in bioactive compounds of pomegranate peel, and we investigated if pomegranate peel extract and fractions would be effective to inhibit lipid and protein oxidation, and discolouration of bluefish patties stored at 4 °C for 9 days, after UV-C irradiation. The non-fractionated SC-CO2 extract from pomegranate peel was rich in phenolic compounds, mainly ellagitannins, besides, it possessed lipophilic compounds such as tocopherols and ß-carotene. These compounds were successfully separated by the fractionation protocols, in a lipid fraction concentrated in lipophilic compounds, and one or two fractions enriched with phenolic compounds, especially ellagitannins. The lipid fraction and the high phenolics fraction from pomegranate peel were then as effective as the synthetic antioxidant BHT in avoiding bluefish patties oxidation during refrigerated storage. Our data indicates that pomegranate peel fractions could be used to replace a synthetic antioxidant in fish meat.

Comprehensive lipid analysis of green Arabica coffee beans by LC-HRMS/MS.

Lipids play an important role in coffee bean development, coffee brew and in the effects of coffee on human health. They account for around 17% of the dry bean weight and encompass different classes and subclasses, mostly triacylglycerols (TAG) and a minor quantity of phospholipids (PL) and ßN-alkanoyl-5-hydroxytryptamides (C-5HT). To comprehensive profile these different lipids, it is important to evaluate extraction methods that provide high lipid coverage and to analyze the lipids in high-resolution techniques. In this work, liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) was employed to comprehensive profile lipids from green Arabica coffee beans and to evaluate the extraction efficiency and lipid coverage of three methods: Bligh-Dyer (BD), Folch (FO), and Matyash (MA). The MA method yielded the greatest number of annotated compounds (131 lipids) compared to the other methods. In the positive electrospray ionization (ESI) mode, the main difference among extraction methods was observed for TAG and diacylglycerols, whereas for the negative ESI it was observed differences for phosphatidylinositol (PI), lysophosphatidylinositol and phosphatidic acid (p < 0.05). The analysis of coffees from different maturation stages and/or post-harvest processes were also performed using the MA method. Immature beans were discriminated from mature and overripe beans by its lower levels of C-5HT, PI, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, and lysophosphatidylethanolamine. These results can help to better understand the coffee lipid composition and its association with coffee quality.

Comprehensive Metabolome Analysis of Fermented Aqueous Extracts of Viscum album L. by Liquid Chromatography-High Resolution Tandem Mass Spectrometry.

Fermented aqueous extracts of Viscum album L. are widely used for cancer treatment in complementary medicine. The high molecular weight compounds viscotoxins and lectins are considered to be the main active substances in the extracts. However, a vast number of small molecules (≤1500 Da) is also expected to be present, and few studies have investigated their identities. In this study, a comprehensive metabolome analysis of samples of fermented aqueous extracts of V. album from two host tree species (Malus domestica and Pinus sylvestris), both prepared by two pharmaceutical manufacturing processes, was performed by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). A total of 212 metabolites were putatively annotated, including primary metabolites (e.g., amino acids, organic acids, etc.) and secondary metabolites (mostly phenolic compounds). A clear separation between V. album samples according to the host tree species, but not due to manufacturing processes, was observed by principal component analysis. The biomarkers responsible for this discrimination were assessed by partial least squares-discriminant analysis. Because V. album extracts from different host trees have different clinical applications, the present work highlights the possibility of characterizing the metabolome for identification and traceability of V. album fermented aqueous extracts.