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.

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.

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

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.

The plant Stryphnodendron adstringens (Mart.) Coville as a neutralizing source against some toxic activities of Bothrops jararacussu snake venom.

Snakebites produce several toxic effects in victims, such as hemorrhage, tissue necrosis, hemostatic, renal, or cardiotoxic alterations, inflammation, and death. To counteract these symptoms, antivenom is the official treatment. Although such therapy prevents death, it does not efficiently neutralize necrosis or other local effects, leading to amputation or morbidities of the affected limb. Therefore, the search for better and more efficient therapies deserves attention; further, plants have been used to ameliorate a number of diseases and medical conditions, including snakebites, for many years. Thus, the aim of this work was to evaluate the antivenom effect of the crude extract, fractions (aqueous and diethyl acetate), and subfractions derived from the aqueous fraction (P1, P2, P3, and P4) of the plant Stryphnodendron adstringens against in vitro (coagulation and proteolytic) and in vivo (edema, hemorrhage, and myotoxic) activities caused by Bothrops jararacussu venom. Overall, all extracts inhibited the toxic effect of B. jararacussu venom, but with different potencies, regardless of whether plant samples were incubated together with venom or injected before or after venom injection into animals; the crude extract and aqueous fraction were found to be the most effective. Indeed, phytochemical and mass spectrometry analysis of S. adstringens samples revealed the presence of flavonols, tannins, and saponins. In conclusion, the plant S. adstringens may represent a promising natural source of molecules to treat the toxic effects associated with envenomation by B. jararacussu snakebites.

Phytochemical analysis and in vitro anti-proliferative activity of Viscum album ethanolic extracts.

BACKGROUND: Viscum album L. (Santalaceae), commonly known as mistletoe, is a hemiparasitic plant traditionally used in complementary cancer treatment. Its antitumor potential is mostly attributed to the presence of aqueous soluble metabolites; however, the use of ethanol as solvent also permits the extraction of pharmacological compounds with antitumor potential. The clinical efficacy of mistletoe therapy inspired the present work, which focuses on ethanolic extracts (V. album "mother tinctures", MT) prepared from different host trees. METHODS: Samples from three European subspecies (album, austriacum, and abietis) were harvested, and five different V. album-MT strains were prepared. The following phytochemical analyses were performed: thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and liquid chromatography-high resolution mass spectrometry (LC-HRMS). The proliferation assay was performed with WST-1 after incubation of tumor (Yoshida and Molt-4) and fibroblast cell lines (NIH/3 T3) with different MT concentrations (0.5 to 0.05% v/v). The cell death mechanism was investigated by flow cytometry (FACS) using Annexin V-7AAD. RESULTS: Chemical analyses of MT showed the presence of phenolic acids, flavonoids and lignans. The MT flavonoid and viscotoxin contents (mg/g fresh weight) were highest in Quercus robur (9.67 ± 0.85 mg/g) and Malus domestica (3.95 ± 0.58 mg/mg), respectively. The viscotoxin isoform proportions (% total) were also different among the VA subspecies with a higher content of A3 in V. album growing on Abies alba (60.57 ± 2.13). The phytochemical compounds as well as the viscotoxin contents are probably related to the antitumor effects of MT. The cell death mechanisms evaluated by colorimetric and FACS methodologies involved necrotic damage, which was host tree-, time- and dose- dependent, with different selectivity to tumor cells. Mother tincture from V. album ssp. abietis was the most effective at inducing in vitro cellular effects, even when incubated at the smallest concentration tested, probably because of the higher content of VT A3. CONCLUSION: Our results indicate the promising antitumor potential of Viscum album ethanolic extracts and the importance of botanical and phytochemical characterization for in vitro anti-proliferative effects.

Production of bioactive films of carboxymethyl cellulose enriched with green coffee oil and its residues.

Carboxymethyl cellulose (CMC)-based films were developed by incorporating green coffee oil (GCO) obtained by cold pressing and hydroalcoholic extracts of its residues. The effect of cake (CE) and sediment extracts (SE) in different proportions (20-40%) and GCO on chemical, morphological, physical, mechanical, optical, and antioxidant properties of the films was investigated. Eight fatty acids and four major phenolic compounds were identified by High-Resolution Direct-Infusion Mass Spectrometry in GCO and residue extracts. FTIR indicated interactions among CMC, phenolic compounds, and fatty acids. Films enriched with residue extracts presented heterogeneous microstructure. The tensile strength of the films decreased from 58 to 3 MPa with the extracts concentration, while elongation increased from 28 to 156% (p < 0.05). The water vapor permeability (averaging 3.94 × 10-8 g mm/cm2 h Pa) was not significantly affected by the extracts and GCO. The surface color was influenced by the type and concentration of extracts (p < 0.05), the film with SE40% had remarkable UV-vis barrier properties. The incorporation of GCO residue extracts imparted high antioxidant capacity to the CMC-based films, especially with CE40% (643.8 µmol Trolox eq./g dried film; 51.3 mg GAE/g dried film). General observations indicated the potential of these films, mainly the ones containing CE, like active packaging material for food applications.

Libidibia ferrea (jucá), a Traditional Anti-Inflammatory: A Study of Acute Toxicity in Adult and Embryos Zebrafish (Danio rerio).

The plant species Libidibia ferrea (Mart. ex Tul.) LP Queiroz var. ferrea basionym of Caesalpinia ferrea (Mart. ex Tul.) is used in various regions of Brazil in folk medicine in the treatment of several health problems, especially in acute and chronic inflammatory processes. Most of the preparations employed are alcoholic. Therefore, this study aimed to evaluate the acute toxicity of the hydroethanolic extract of fruits of Libidibia ferrea (EHEFLf) in zebrafish, emphasizing the possible changes in the organic-cellular level of the gills, liver, kidneys, and intestine and on embryos. The result obtained by LC-M/MS from EHEFLf indicated a high concentration of possible polyhydroxylated substances. EHEFLf, at a dose of 2 g/kg orally, produced non-significant alterations of the analyzed organs. However, for embryos, the treatment with different concentrations demonstrated heart toxicity that was concentration-dependent. There is no evidence of a correlation of the observed effects with the phytochemical composition, and considering the species of animal used, it can be suggested that the oral use of L. ferrea hydroethanolic extract has an acceptable degree of safety for use as an oral medicinal product. and embryo results have shown significant affinity to the heart; however, it is perceived to be related to the concentrations used.

Extending compound identification for molecular network using the LipidXplorer database independent method: A proof of concept using glycoalkaloids from Solanum pseudoquina A. St.-Hil.

INTRODUCTION: Molecular networks are now established as the method of choice for tandem mass spectrometry dereplication and similarity-based structure elucidation. Node identification can be used to start the propagation of the structure elucidation of unknown compounds progressively. OBJECTIVE: To demonstrate the capabilities of using the LipidXplorer data results along with molecular networking to identify nodes and aid sequential structure elucidation of unknown compounds. MATERIAL AND METHODS: Molecular fragmentation query language (MFQL) files were written to identify glycoalkaloids based on known structures described for Solanum species. A dataset generated from liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis of Solanum pseudoquina sample were submitted to dereplication on both LipidXplorer software and Global Natural Products Social Molecular Network (GNPS) online system. The resulting attribute table from GNPS calculations was merged with the LipidXplorer results and this merged file was used for network visualisation in Cytoscape. Nodes in the molecular network were labelled using the LipidXplorer identifiers, thus assisting the structure elucidation of unidentified compounds. RESULTS: The combination of the LipidXplorer glycoalkaloids list and GNPS analysis was used in Cytoscape to label nodes in the molecular network. The analysis of the network using these labelled starting points triggered the structure elucidation of closely related nodes leading to the identification of 30 compounds using the LipidXplorer output and four purified and structure elucidated compounds, including a new glycoalkaloids identified as 3-O-(ß-d-xylopyranosyl)-(20R,25S)-22,26-epimino-16-acetyl-cholesta-5,22(N)-diene. CONCLUSION: A significant compound identification completely based on molecular formula and fragmentation queries was achieved. This new and effective approach could help researches to expand the identification rate of compounds in dereplication studies using molecular networks.

Protective Effect of the Plant Extracts of Erythroxylum sp. against Toxic Effects Induced by the Venom of Lachesis muta Snake.

Snake venoms are composed of a complex mixture of active proteins that induce toxic effects, such as edema, hemorrhage, and death. Lachesis muta has the highest lethality indices in Brazil. In most cases, antivenom fails to neutralize local effects, leading to disabilities in victims. Thus, alternative treatments are under investigation, and plant extracts are promising candidates. The objective of this work was to investigate the ability of crude extracts, fractions, or isolated products of Erythroxylum ovalifolium and Erythroxylum subsessile to neutralize some toxic effects of L. muta venom. All samples were mixed with L. muta venom, then in vivo (hemorrhage and edema) and in vitro (proteolysis, coagulation, and hemolysis) assays were performed. Overall, crude extracts or fractions of Erythroxylum spp. inhibited (20%-100%) toxic effects of the venom, but products achieved an inhibition of 4%-30%. However, when venom was injected into mice before the plant extracts, hemorrhage and edema were not inhibited by the samples. On the other hand, an inhibition of 5%-40% was obtained when extracts or products were given before venom injection. These results indicate that the extracts or products of Erythroxylum spp. could be a promising source of molecules able to treat local toxic effects of envenomation by L. muta venom, aiding in the development of new strategies for antivenom treatment.

Arabica and robusta coffees: identification of major polar compounds and quantification of blends by direct-infusion electrospray ionization-mass spectrometry.

Considering that illegal admixture of robusta coffee into high-quality arabica coffee is an important task in coffee analysis, we evaluated the use of direct-infusion electrospray ionization-mass spectrometry (ESI-MS) data combined with the partial least-squares (PLS) multivariate calibration technique as a fast way to detect and quantify arabica coffee adulterations by robusta coffee. A total of 16 PLS models were built using ESI± quadrupole time-of-flight (QTOF) and ESI± Fourier transform ion cyclotron resonance (FT-ICR) MS data from hot aqueous extracts of certified coffee samples. The model using the 30 more abundant ions detected by ES+ FT-ICR MS produced the most accurate coffee blend percentage prediction, and thus, it was later successfully employed to predict the blend composition of commercial robusta and arabica coffee. In addition, ESI± FT-ICR MS analysis allowed for the identification of 22 compounds in the arabica coffee and 20 compounds in the robusta coffee, mostly phenolics.