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.

Dynamics of volatile compounds in TSH 565 cocoa clone fermentation and their role on chocolate flavor in Southeast Brazil.

The effects of indigenous fermentation on volatile compound profiles in a Theobroma cacao L, TSH565 clone, resistant to Moniliophtora perniciosa and Phytophthora spp. were evaluated in Southern Brazil. Sixty-three volatile flavor compounds in pulp and 36 in grains were identified by SPME-HS/GC-MS and classified as terpenes, alcohols, esters, ketones and aldehydes, among others. The relative amount of these compounds and their evolution until the end of the fermentation process were assessed in both fresh and fermented grains/pulp masses. ß-myrcene and ß-cis-ocimene, among terpenes, were detected in high amounts and are associated to a fine chocolate aroma. The sensory evaluation of chocolates manufactured from the fermented cocoa was performed by trained panelists, which defined 15 sensory descriptors. Chocolates from the TSH565 cultivar were characterized by a rich, fruity, intense cocoa flavor and bitterness, which are valuable sensorial and commercial attributes.

Gas chromatography-mass spectrometry of sapucainha oil (Carpotroche brasiliensis) triacylglycerols comprising straight chain and cyclic fatty acids.

Sapucainha oil, which may be used to treat leprosy, comprises straight chain and cyclic fatty acids (FA), and triacylglycerols (TAG). The FA and TAG content of the oil sample was analysed using gas chromatography-electron ionisation mass spectrometry (GC-EIMS). FA analysis was performed after derivatisation to fatty acid methyl esters (FAME). For free FA and TAG analysis, the oil sample was dissolved in hexane and injected into a short, high-temperature column, for GC with MS analysis. Free FA and FAME were tentatively identified based on mass spectrum information of their molecular and fragment ions, as well as library matching. Overlapping TAG peaks were deconvoluted based on mass fingerprint data. The FA composition was utilised to predict possible TAG identities. FA residues of TAG were identified based on characteristic fragment ions, such as [M-RCO2]+, [RCO+128]+, [RCO+74]+ and RCO+ where R is the aliphatic hydrocarbon chain. FAME analysis showed that the cyclic FA hydnocarpic (36.1%), chaulmoogric (26.5%) and gorlic (23.6%) acids were the major components. In addition, straight chain FA such as palmitic, palmitoleic, stearic, oleic and linoleic acids were detected. Palmitic, oleic, hydnocarpic, chaulmoogric and gorlic acids were also detected as free FA in the oil sample. Six groups of TAG peaks were eluted from GC at temperatures ≥330 °C. After deconvolution and mass spectrum analysis, each TAG peak group was revealed to comprise 2 to 5 co-eluted TAG molecules; >18 TAG were identified. These TAG consisted of a mix of both cyclic and straight chain FA, but were mostly derived from cyclic FA.

Reduction of ßN-alkanoyl-5-hydroxytryptamides and diterpenes by yeast supplementation to green coffee during wet processing.

Coffee is one of the most consumed non-alcoholic beverages in the world. It is well known that some compounds present in coffee beans have important biological activities. In this study, evidence was turned to ßN-alkanoyl-5-hydroxytryptamides (C-5HTs) and to the furokaurane diterpenes cafestol and kahweol, associated with gastric irritation and increasing of blood cholesterol, respectively. Fermentation in coffee post-harvest wet process was induced by three Saccharomyces cerevisiae yeasts (for bakery, white and sparkling wines) as starter cultures. Variations in mass, time, temperature and pH (56 experiments under fractional factorial and mixture experimental designs) were tested. Substantial reductions for C-5HTs (up to 38% reduction for C20-5HT and 26% for C22-5HT) as well as for diterpenes (54% for cafestol and 53% for kahweol) were obtained after treating green coffee beans with 0.6 g of a 1:1:1 mixture the three yeasts for 12 h at 15 °C and pH 4. Caffeine and 5-CQA content, monitored in the green coffee beans, did not change. Therefore, the use of starter cultures during coffee post-harvest wet process has influence on the amount of some important compounds related to health and improves the sensory quality of the beverage.

Ionic liquid capillary columns for analysis of multi-component volatiles by gas chromatography-mass spectrometry: performance, selectivity, activity and retention indices.

This study investigates applications and performance evaluation of SLB-IL60, SLB-IL76 and SLB-IL111 columns, in relation to a DB-Wax column, for the analysis of coffee volatile compounds. Both standards and an authentic coffee sample were analysed, with solid-phase microextraction (SPME) sampling of the latter. A cryofocusing method was applied to improve resolution of the earliest eluting peaks using splitless injection SPME sample analysis. The Grob test was used to verify the inertness and efficiency of the columns, helping to understand the interactions between analytes and stationary phases, particularly toward more polar coffee analytes. A DB-5 column was used only in analysis of n-alkanes and Grob test mixtures as an apolar reference. The evaluated ionic liquid columns showed a moderate acid-base character and low inertness for compounds with hydrogen bond capabilities, especially for the hydroxylated analytes, 2,3-butanediol in the Grob test, and furanones and acids in the coffee standards. The columns exhibiting the best resolution and efficiency were DB-Wax and SLB-IL60, both for samples and standards. Although the DB-Wax column is preferred for analysis of coffee volatiles, due to better inertness, the evaluated ionic liquid columns allowed identification of compounds that were not observed in separations with the Wax-phase column in this work. Among these compounds is 3,4-dimethyl-2,5-furandione, seldom reported in the literature of coffee. Proposed improvement by the manufacturer in the inertness of the columns evaluated in this study may lead to better results; so future versions of IL phases may be better applied in the separation of target analytes, especially those with basic character. Graphical abstract Illustrative representation of the sample (coffee) and the chemical structures of the stationary phases of the ionic liquid capillary columns used as object of study in the present work.

Studies on the volatile fraction composition of three native Amazonian-Brazilian fruits: Murici (Byrsonima crassifolia L., Malpighiaceae), bacuri (Platonia insignis M., Clusiaceae), and sapodilla (Manilkara sapota L., Sapotaceae).

The volatile fraction of murici, bacuri and sapodilla are here studied because of their increasing interest for consumers, abundance of production in Brazil, and the general demand for new flavors and aromas. Their volatile profiles were studied by two High Concentration Capacity Headspace techniques (HCC-HS), Headspace Solid Phase Microextraction (HS-SPME) and Headspace Sorptive Extraction (HSSE), in combination with GC-MS. Murici volatile fraction mainly contains esters (38%), carboxylic acids (19%), aldehydes (11%), alcohols (14%), others (13%) and sulfur compounds; bacuri is characterized by terpenes (41%), non-terpenic alcohols (24%), esters (15%), aldehydes (6%), and others (12%); sapodilla consists of esters (33%), alcohols (27%), terpenes (18%) and others (21%). The GC-MS component co-elution was overcome by GC×GC-qMS. The adoption of modern analysis technologies afforded to achieve a better knowledge of the volatile fraction composition of these fruit pulps by increasing substantially the number of compounds identified.

Qualitative analysis of Copaifera oleoresin using comprehensive two-dimensional gas chromatography and gas chromatography with classical and cold electron ionisation mass spectrometry.

Improved separation of both sesquiterpenes and diterpenic acids in Copaifera multijuga Hayne oleoresin, is demonstrated by using comprehensive two-dimensional gas chromatography (GC×GC) coupled to accurate mass time-of-flight mass spectrometry (accTOFMS). GC×GC separation employs polar phases (including ionic liquid phases) as the first dimension (1D) column, combined with a lower polarity 2D phase. Elution temperatures (Te) of diterpenic acids (in methyl ester form, DAME) increased as the 1D McReynolds' polarity value of the column phase decreased. Since Te of sesquiterpene hydrocarbons decreased with increased polarity, the very polar SLB-IL111 1D phase leads to excessive peak broadening in the 2D apolar phase due to increased second dimension retention (2tR). The combination of SLB-IL59 with a nonpolar column phase was selected, providing reasonable separation and low Te for sesquiterpenes and DAME, compared to other tested column sets, without excessive 2tR. Identities of DAME were aided by both soft (30eV) electron ionisation (EI) accurate mass TOFMS analysis and supersonic molecular beam ionisation (cold EI) TOFMS, both which providing less fragmentation and increased relative abundance of molecular ions. The inter-relation between EI energies, emission current, signal-to-noise and mass error for the accurate mass measurement of DAME are reported. These approaches can be used as a basis for conducting of GC×GC with soft EI accurate mass measurement of terpenes, particularly for unknown phytochemicals.

Structure Elucidation, Antimicrobial and Cytotoxic Activities of a Halimane Isolated from Vellozia kolbekii Alves (Velloziaceae).

A new halimane diterpene was isolated from Vellozia kolbekii Alves (Velloziaceae) and identified as (5R,8R,9S,13R)-halim-1,10-ene-15,16-diol (1). It showed cytotoxicity against three human cancer cell lines, SF-295 (glioblastoma), MDA-MB-435 (melanoma), and HCT-8 (colon adenocarcinoma). In the mechanism of cytotoxic action, halimane 1 interferes in two major phases of the cell cycle: in S phase, in which DNA synthesis occurs and where it is very sensitive to damage, and G2M phase which is the phase of preparation for mitosis and mitosis itself, showing apoptosis-inducing properties. Antimicrobial activity towards Gram-positive and Gram-negative bacteria was studied and, against Bacillus cereus, B. subtilis, Escherichia coli, and Pseudomonas aeruginosa, a MIC value of 0.025 µM was observed for halimane 1, which is more active than the positive control chloramphenicol.

Interactions between irrigants commonly used in endodontic practice: a chemical analysis.

INTRODUCTION: The aim of this work was to characterize the by-products formed in the associations between the most commonly used irrigants in endodontic practice through electrospray ionization quadrupole time-of-flight mass spectrometry analyses. METHODS: Sodium hypochlorite (NaOCl) (0.16%, 1%, 2.5%, and 5.25%) was associated with 2% chlorhexidine (CHX) solution and gel, 17% EDTA, 10% citric acid, 37% phosphoric acid, saline solution, ethanol, and distilled water. CHX solution and gel were also associated with all above mentioned irrigants. The solutions were mixed in a 1:1 ratio, and electrospray ionization quadrupole time-of-flight mass spectrometry was used to characterize the precipitates when formed. RESULTS: CHX produced an orange-brown precipitate when associated with NaOCl from 1%-5.25% and an orange-white precipitate when associated with 0.16% NaOCl. When associated with EDTA, CHX produced a white milky precipitate, and when associated with saline solution and ethanol, a salt precipitation was produced. No precipitation was observed when CHX was associated with citric acid, phosphoric acid, or distilled water. In the NaOCl associations, precipitation occurred only when CHX was present. CONCLUSION: The orange-brown precipitate observed in the association between CHX and NaOCl occurs because of the presence of NaOCl, an oxidizing agent causing chlorination of the guanidino nitrogens of the CHX. The precipitates formed in the reaction of CHX with EDTA, saline solution, and ethanol were associated with acid-base reactions, salting-out process, and lower solubility, respectively. NaOCl associated with EDTA, citric acid, and phosphoric acid leads mainly to chlorine gas formation. Intermediate flushes with distilled water seem to be appropriate to prevent or at least reduce formation of by-products.

Chemical composition and biological activities of soldiers of the Brazilian termite species, Nasutitermes macrocephalus (Isoptera: Natutitermitinae).

The defensive secretion of the frontal gland from termite soldiers is a mixture of monoterpenes, sesquiterpenes and diterpenes, the latter being the most representative. Analyses of the dichloromethane extract from soldiers of the Brazilian termite, Nasutitermes macrocephalus (Silvestri, 1903) (Isoptera, Nasutitermitinae), described for the first time, allowed to identify the presence of two monoterpenes (alpha-pinene and limonene) and two sesquiterpenes (beta-trans-caryophyllene and gamma-selinene) [corrected] by GC-EIMS, and the isolation of one rippertane and six trinervitane diterpenes by RP-HPLC. The chemical structures of the purified compounds were elucidated by interpretation of their spectroscopic data (1D and 2D NMR, EIMS, HRESIMS, and specific optical rotation) and the complete unequivocal assignment of the 3a-hydroxy-trinervita-1(15),8(19)-dien-2-one (6) was included in this paper, to complement the lack of information in the literature. Antibacterial, antifungal and cytotoxicity against cancer cell lines activities were evaluated. In particular, the compounds 2alpha,3beta-dihydroxytrinervita-l(15),8(19)-diene (2) and 3alpha-hydroxy-15-rippertene (7) exhibited the better activities against the clinically isolated Gram-positive bacterium methicillin-resistant Staphylococcus aureus BMB 9393, both with a MIC value of 31.2 microg mL(-1). This is the first description of a rippertane diterpene (7) as an antibacterial agent.

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.

Synthesis and analgesic profile of conformationally constrained N-acylhydrazone analogues: discovery of novel N-arylideneamino quinazolin-4(3H)-one compounds derived from natural safrole.

In this work we reported the synthesis and evaluation of the analgesic, anti-inflammatory, and platelet anti-aggregating properties of new 3-(arylideneamino)-2-methyl-6,7-methylenedioxy-quinazolin-4(3H)-one derivatives (3a-j), designed as conformationally constrained analogues of analgesic 1,3-benzodioxolyl-N-acylhydrazones (1) previously developed at LASSBio. Target compounds were synthesized in very good yields exploiting abundant Brazilian natural product safrole (2) as starting material. The pharmacological assays lead us to identify compounds LASSBio-1240 (3b) and LASSBio-1272 (3d) as new analgesic prototypes, presenting an antinociceptive profile more potent and effective than dipyrone and indomethacin used, respectively, as standards in AcOH-induced abdominal constrictions assay and in the formalin test. These results confirmed the success in the exploitation of conformation restriction strategy for identification of novel cyclic N-acylhydrazone analogues with optimized analgesic profile.

Antinociceptive and hypothermic evaluation of the leaf essential oil and isolated terpenoids from Eugenia uniflora L. (Brazilian Pitanga).

Eugenia uniflora L. (Myrtaceae), known as Brazilian cherry tree, is a fruity tree spread all over Brazil used in popular medicine to treat inflammations, rheumatic pain and fever, as hypoglycemic, diuretic and has been widely used in the cosmetics industry. The present study discusses the chemical composition, the antinociceptive and hypothermic profile of the essential oil of pitangueira leaves. The chemical composition was evaluated by GC-MS and the main constituent of the oil was characterized, after isolation, as a mixture of atractylone (1) and 3-furanoeudesmene (2). The essential oil, its pentane fraction and the isolated mixture of sesquiterpenes (1 and 2), given orally, significantly inhibited the acetic acid-induced abdominal constrictions, increased the latency time in hot plate test and showed a hypothermic effect. The results suggest that the responsible for the antinociceptive and hypothermic effect were the isolated furanosesquiterpenes. These findings provided additional pharmacological information and may contribute for the use of Brazilian cherry tree as a phytomedicine.

Two fast screening methods (GC-MS and TLC-ChEI assay) for rapid evaluation of potential anticholinesterasic indole alkaloids in complex mixtures.

The pharmacotherapy for Alzheimer's disease (AD) includes the use of acetylcholinesterase inhibitors (AChEI). Recent investigations for novel AD therapeutic agents from plants suggested that Tabernaemontana genus is a promising source of novel anticholinesterasic indole alkaloids. In this work two fast screening techniques were combined in order to easily identify novel cholinesterase inhibitors (ChEI). Gas chromatography-mass spectrometry (GC-MS) of the less polar alkaloidic fractions obtained from the acid-base extraction of the stalk of T. laeta revealed thirteen monoindole alkaloids, four of them confirmed by co-injection with previously isolated alkaloids. The others were tentatively identified by mass fragmentation analysis. By gas chromatography with flame ionization detection (GC-FID) and using isatin as internal standard, affinisine and voachalotine were determined as major compounds. These fractions and fourteen previously isolated alkaloids, obtained from root bark of T. laeta and T. hystrix were investigated for acetyl (AChE) and butyrylcholinesterase (BuChE) inhibitory activities by the modified Ellman's method in thin layer chromatography(TLC-ChEI). Results showed selective inhibition of the alkaloids heyneanine and Nb-methylvoachalotine for BuChE, and 19-epi-isovoacristine for AChE, whereas olivacine, affinisine, ibogamine, affinine, conodurine and hystrixnine inhibited both enzymes. In addition to confirming that monoterpenoid indole alkaloids can be novel therapeutic agents for AD, this is the first report of the ChEI activity of olivacine, a pyridocarbazole alkaloid.

Two fast screening methods (GC-MS and TLC-ChEI assay) for rapid evaluation of potential anticholinesterasic indole alkaloids in complex mixtures

The pharmacotherapyfor Alzheimer's disease (AD) includes the use of acetylcholinesterase inhibitors (AChEI). Recent investigations for novel AD therapeutic agents from plants suggested that Tabernaemontana genus is a promising source of novel anticholinesterasic indole alkaloids. In this work two fast screening techniques were combined in order to easily identify novel cholinesterase inhibitors (ChEI). Gas chromatography-mass spectrometry (GC-MS) of the less polar alkaloidic fractions obtained from the acid-base extraction of the stalk of T. laeta revealed thirteen monoindole alkaloids, four of them confirmed by co-injection with previously isolated alkaloids. The others were tentatively identified by mass fragmentation analysis. By gas chromatography with flame ionization detection (GC-FID) and using isatin as internal standard, affinisine and voachalotine were determined as major compounds. These fractions and fourteen previously isolated alkaloids, obtained from root bark of T. laeta and T. hystrix were investigated for acetyl (AChE) and butyrylcholinesterase (BuChE) inhibitory activities by the modified Ellman's method in thin layer chromatography(TLC-ChEI). Results showed selective inhibition of the alkaloids heyneanine and Nb-methylvoachalotine for BuChE, and 19-epi-isovoacristine for AChE, whereas olivacine, affinisine, ibogamine, affinine, conodurine and hystrixnineinhibited both enzymes. In addition to confirming that monoterpenoid indole alkaloids can be novel therapeutic agents for AD, this is the first report of the ChEI activity of olivacine, a pyridocarbazole alkaloid.

Dentre os tratamentos da doença de Alzheimer (DA) está o uso de inibidores da enzima acetilcolinesterase. Pesquisas recentes visando a descoberta de novos agentes terapêuticos naturais para esta doença sugerem que o gênero Tabernaemontana é uma fonte promissora de alcalóides indólicos anticolinesterásicos. Neste trabalho, duas técnicas de análise em mistura foram associadas de modo a identificar facilmente novos inibidores colinesterásicos. A cromatografia em fase gasosa acoplada a espectrometria de massas (CG-EM) das frações alcaloídicas apolares, obtidas da extração ácido-base do caule de T. laeta, revelou a presença de treze alcalóides monoindólicos, quatro deles confirmados por co-injeção com padrões previamente isolados. Os outros alcalóides foram tentativamente identificados pelo padrão de fragmentação de massas. Por cromatografia em fase gasosa com detecção por ionização de chama (CG-DIC) e utilizando isatina como padrão interno, affinisina e voachalotina foram identificadas como substâncias majoritárias. As frações alcaloídicas obtidas e os quatorze alcalóides previamente isolados das raízes de T. laeta e T. hystrix foram analisados quanto à atividade inibitória das enzimas acetil (AChE) e butirilcolinesterase (BuChE) pelo método de Ellman em cromatografia em camada delgada (CCD-ChEI). Os resultados revelaram uma inibição seletiva dos alcalóides heyneanina e Nb-metilvoachalotina para BuChE e de 19-epi-isovoacristina para AChE, enquanto que olivacina, affinisina, ibogamina, affinina, conodurina e hystrixnina inibiram ambas as enzimas. Além de confirmar que alcalóides indólicos monoterpênicos são agentes terapêuticos promissores para o tratamento da DA, este é o primeiro relato da atividade anticolinesterásica de olivacina, um alcalóide piridocarbazólico.

Indole alkaloids from Tabernaemontana australis (Muell. Arg) Miers that inhibit acetylcholinesterase enzyme.

Ten indole alkaloids from the chloroform extract of stalk of Tabernaemontana australis (Muell. Arg) Miers were tentatively identified by GC-MS, viz., coronaridine (1), voacangine (2), voacangine hydroxyindolenine (3), rupicoline (4), ibogamine (5), ibogaine (6), ibogaline (7), desethyl-voacangine (8), voachalotine (9), and affinisine (10). Of these, the first four were isolated by silica gel open column chromatography, identified by uni- and bidimensional NMR, IR, MS and showed anti-cholinesterasic activity at the same concentration as the reference compounds physostigmine and galanthamine (detection limit of 0.01mM) by TLC assay using the modified Ellman's method.

Two xanthones from Polygala paniculata and confirmation of the 1-hydroxy-2,3,5-trimethoxy-xanthone at trace level by HRGC-MS.

Polygala paniculata L. yielded the xanthones 1-hydroxy-5-methoxy-2,3-methylenedioxyxanthone (1) and 1,5-dihydroxy-2,3-dimethoxyxanthone (2), together with coumarin murragatin and flavonol rutin. Their structures were established by chemical and spectroscopic methods (EIMS, IR, 1H and 13C NMR, NOE difference spectroscopy). By posterior analysis of an apolar crude extract using high resolution gas chromatography coupled to mass spectrometry (HRGC-MS) it was possible to characterize two sterol (spinasterol and delta25-spinasterol) and the minor 1-hydroxy-2,3,5-trimethoxyxanthone (3). Thus, the xanthone 3 was confirmed through of co-injection HRGC-MS of the respective extract with a certified standard obtained by methylation of 2 with diazomethane.