Penicillium meliponae, a recently described and rare species, was isolated as an endophytic fungus from the Amazonian plant Duguetia sthelechantha, and has been proven to be a pigment producer. Considering the high productivity of this species and the lack of data on its chemical composition, the present study aimed to characterize the chemical profile of P. meliponae and evaluate the influence of agitation and the use of different culture media. For this purpose, liquid chromatography coupled with mass spectrometry (LC-MS/MS) and molecular networking were used, allowing the identification of 17 azaphilone molecules with sclerotiorin-like skeletons, becoming the first chemical report of this species. In addition, the different production patterns in the tested culture media were indicative that this species is sensitive to changes in the composition of the carbon source and to the presence of agitation. Furthermore, this work contributes to the fragmentation mechanisms of the different possible structural arrangements for azaphilones of the sclerotiorin type and serves as a repository of information on the gas-phase behavior of this type of metabolite in mass spectrometry experiments and will assist future studies aimed at the discovery of azaphilones.

Ambuic acid (AA) is a highly-modified cyclohexenone and known as a promising inhibitor of quorum sensing in methicillin-resistant Staphylococcus aureus, and is thus a candidate as an antivirulence drug. This molecule is mainly produced by the species of Pestalotiopsis and, since its discovery twenty years ago, only a restricted amount of AA-derivatives have been described. Despite being a promising subject, methods for the analysis of modified AA-analogues via mass spectrometry remain unexplored. In order to adress this question, the marine fungus Pestalotiopsis sp. 4A11 associated with the ascidian Didemnum perlucidum was grown in a solid rice medium and its crude extract was chemically studied. From this extract, AA and 10-hydroxy ambuic acid (10-HAA) were isolated and identified using NMR spectroscopy with the aim of obtaining model compounds for the MS analysis. These served as reference compounds (seeds) to guide the dereplication of other AA-analogues via LC-MS/MS-based molecular networking. Based on the manual interpretation of the fragmentation pathways of the seeds and related compounds observed in the networks, six AA-derivatives were dereplicated in the extract. Furthermore, three analogues with unprecedented chemical formulas were proposed as putative unprecedented AA-derivatives. The fragmentation annotation proposed represents a fast and feasible method for characterizing AA-derivatives.

The phytochemical investigation of the hexane and methanol extracts from the bark of Duguetia pycnastera Sandwith (Annonaceae) afforded seven known compounds, two asarone-derived phenylpropanoids and five isoquinoline-derived alkaloids. The asarones, γ-asarone (1-allyl-2,4,5-trimethoxybenzene) and 2,4,5-trimethoxy-styrene were isolated of the hexane extract while the aporphine alkaloids, O-methylmoschatoline, lysicamine, nornuciferidine, and guatterine N-oxide, and the benzyltetrahydroisoquinoline alkaloid, (S)-reticuline were isolated of the alkaloid fraction of the methanol extract. This is the first report of these compounds in D. pycnastera. γ-Asarone is being reported for the first time in the Annonaceae. Nornuciferidine is described for the second time in the Annonaceae while guatterine N-oxide is the third register. The structures of the isolated compounds were established by extensive analyses using 1D and 2D NMR spectroscopy in combination with MS. The cytotoxic activity of the isolated compounds (except for nornuciferidine) was evaluated against cancer and non-cancerous cell lines, in which lysicamine was the most active compound, mainly against HL-60, HepG2, and K562 with IC50 values of 24.40, 28.86 and 38.75 µmol L-1, respectively.

The phytochemical investigation of the hexane extract which was obtained from the leaves of Xylopia laevigata (Annonaceae) produced six terpenes. Among these, five diterpenes named abieta-7,13-dien-3-one, ent-7β-acetoxy-16β-hydroxy-kaurane, 4-epi-cupressic acid, powerol, and labdorffianic acid B, and one oxygenated sesquiterpene, spathulenol. This is the first time these isolated diterpenes from X. laevigata have been described. The different structures belong to the abietane, kaurane, and labdane diterpenoids, and are indicative of the chemical diversity found in X. laevigata. Moreover, the diterpene ent-7β-acetoxy-16β-hydroxy-kaurane is reported herein for the first time as a natural product, and 4-epi-cupressic acid, powerol, and labdorffianic acid B are reported for the first time in the Annonaceae family. The structures of the isolated compounds were established by extensive analyses using 1D and 2D NMR spectroscopy in combination with MS. The cytotoxic activities of compounds abieta-7,13-dien-3-one, spathulenol, 4-epi-cupressic acid, and powerol were evaluated against tumor and non-tumor cell lines, in which spathulenol was found to be the most active, mainly against K562 with an IC50 value of 17.20 μmol L-1.

Onychopetalum is an Amazonian botanical genus close-related with Bocageopsis and Unonopsis, whose species are used in traditional medicine. Although these genera are widely investigated from the phytochemical viewpoint, the knowledge about Onychopetalum remains almost unexplored. In order to perform a comprehensive characterization of isoquinoline-derived alkaloids from Onychopetalum amazonicum, an integrative approach based on the alkaloid profile analysis by leaf spray mass spectrometry (LS-MS), followed by a robust dereplication through high-performance liquid chromatography coupled with diode array detector and atmospheric pressure chemical ionization tandem mass spectrometry, (HPLC-DAD-APCI-MS/MS) and nuclear magnetic resonance (NMR) analysis was adopted. LS-MS allowed the tentative identification of reticuline, norjuziphine, nornuciferine, O-methylisopiline, nuciferine, N-methyl-O-methylisopiline, anonaine, and N-methylanonaine. HPLC-DAD-MS/MS analysis supported the tentative identification of N-methylasimilobine, asimilobine, norushinsunine, N-methylisopiline, isopiline, liriodenine, and lysicamine. Moreover, the integration of MS and NMR data allowed the additional identification of stepholidine and isocorypalmine. The applied integrative approach proposed herein enabled a comprehensive analysis of the alkaloid content from the O. amazonicum, showing to be a useful strategy for the dereplication of isoquinoline-derived alkaloids.

Bertholletia excelsa Bonpl. (Lecythidaceae) is a South American tree worldwide known for providing the Brazil nuts. In the Amazon Region, B. excelsa is found in monocultures, integrating agroforestries and providing raw materials for food and timber industries. Through the application of an integrative analysis based on high-performance liquid chromatography coupled with diode array detector and tandem mass spectrometry (HPLC-DAD-MS/MS) and nuclear magnetic resonance (NMR) techniques, the present study showed that B. excelsa bark biomass residues contain large quantities of ellagic acid (EA) and its derivatives. Qualitatively, five compounds were characterized for the first time in this species. Quantitations were carried out to determine the total amount of these compounds in outer and inner bark tissues. A total of 4.96 and 44.09 g of EA derivatives per kg of dry residues was determined for the outer and inner barks, respectively. Among the EA derivatives, eschweilenol C, ellagic acid and valoneic acid dilactone were the main compounds. These results pointed B. excelsa barks as a valuable biomass residue with potential to be source of health-promoting compounds. Therefore, a potential raw material as source of valuable bioactive phenolic compounds is described herein.

The continuous search for biologically active compound candidates pushes the pursuit of new substances produced by diverse organisms. Endophytic fungi are known as a promising source of metabolites with several biological activities. Based on the rich Amazonian biodiversity and the chemical potential of microbial sources, three Talaromyces strains, all major endophytes from their respective host plants, were studied aiming at the isolation of biologically active secondary metabolites. Through classical chromatographic approaches, 13 compounds were isolated from the antimicrobial extracts of the studied strains. From these, polyketides, steroids, anhydrides, and phenolic compounds were identified. Among them, two previously undescribed compounds were elucidated based on spectroscopic and spectrometric methods, a homodimer chromanone and a maleic anhydride methyl ester. The antimicrobial activity against a panel of pathogenic microorganisms from extracts, fractions, and isolated compounds was evaluated.

Unonopsis (Annonaceae) is a neotropical genus constituted by nearly fifty species, with fifteen described in Brazil. In the state of Amazonas seven species are found, including U. guatterioides, which displays problems from the botanical viewpoint. Previous studies showed this genus as a promising source of aporphinoid alkaloids. In order to investigate the potential of the leaf alkaloid fingerprint for chemotaxonomic approaches, twelve Unonopsis specimens, representing five species commonly found in the state of Amazonas were subjected to acid-base partitioning to yield the respective alkaloidal fractions. These fractions were analysed by direct infusion electrospray ionization multiple stage mass spectrometry (ESI-MSn). The obtained data were treated through chemometric tools [principal components analysis (PCA) and hierarchical cluster analysis (HCA)]. Multivariate analysis pointed to aporphine, proaporphine and tetrahydroprotoberberine alkaloids as the responsible compounds for segregation of the investigated species, being these alkaloids tentatively identificated by multiple stage mass spectrometry. The alkaloid fingerprint along with multivariate analysis provided a simple and effective approach to differentiateUnonopsis species commonly found in the state of Amazonas.

The continuous search for antimicrobial candidates pushes the pursuit of compounds in the most diverse organisms. Amphibians are known as a prolific source of antibacterial peptides. Based on the rich biodiversity of the Amazon region the unexplored green-tree frog (Hypsiboas cinerascens) was studied for its skin secretion peptide content. Chromatographic separations and established tandem mass spectrometry (MS/MS) methods were used for sequencing the primary structures of the purified compounds. De novo sequencing lead to the identification of five new peptides related to hylaseptin P1, displaying an aminated C-terminal. Sequencing of the complementary deoxyribonucleic acid (cDNA) analysis allowed the disambiguation of isobaric amino-acids for C-01. Matrix assisted laser desorption ionization (MALDI) was carried out, demonstrating the in situ co-occurrence of the identified peptides in the dorsal skin. The major peptide C-01 was synthesized and assayed against a selection of microorganisms displaying minimal inhibitory concentrations (MICs) ranging from 4 to 16 µM.

Buriti (Mauritia flexuosa L. f.) é uma palmeira extensamente distribuída pelas florestas da Amazônia brasileira. Dos frutos do buriti é extraído um óleo que é popularmente utilizado como alimento e com fins medicinais. Neste trabalho reportamos a aplicação de uma abordagem integrativa baseada em espectrometria de massas (MS) utilizando cromatografia a gás acoplada a espectrometria de massas com ionização por elétrons (GC-MS) e espectrometria de massas ambiente com ionização por sonic-spray (EASI-MS) com o objetivo de se obter uma caracterização lipídica abrangente do óleo de buriti. Análises por GC-MS revelaram a presença de diversos ácidos graxos, esteróis além de tocoferóis. EASI-MS permitiu a caracterização de triacilgliceróis e ácidos graxos nos modos positivo e negativo de ionização, respectivamente. Adicionalmente, a capacidade antioxidante foi avaliada para as frações lipofílica e hidrofílica. Os resultados revelaram uma capacidade antioxidante lipofílica (LAC) de 1,8 ± 0,01 e uma hidrofílica de 8,3 ± 0,01 μmol equivalentes de Trolox (TE) g−1, indicando que o óleo de buriti compreende uma valiosa fonte de compostos antioxidantes com potencial em diversas aplicações.

Buriti (Mauritia flexuosa L. f.) is a palm widely distributed in the Amazon Rain Forest of Brazil. Oil is extracted from buriti fruit and popularly used as a food resource and for medicinal purposes. Herein we report the use of an integrative approach based on mass spectrometry (MS) techniques comprising gas chromatography coupled to electron ionization mass spectrometry (GC-MS) and easy ambient sonic-spray ionization mass spectrometry (EASI-MS) with the objective to provide comprehensive lipid characterization of buriti oil. GC-MS analysis revealed the presence of many fatty acids (oleic, palmitic and linoleic), sterols beyond α- and β-tocopherol in buriti oil. EASI-MS allowed the characterization of fatty acids and many triaclyglycerides (TAG) in the negative and positive ion mode, respectively. Additionally, the antioxidant capacity (AC) was measured by the oxygen radical absorbance capacity assay (ORAC); this test was carried with the lipophilic and hydrophilic fractions of the buriti oil. Results revealed a lypophilic antioxidant capacity (LAC) of 1.8 ± 0.01 and hydrophilic antioxidant capacity (HAC) of 8.3 ± 0.01 μmol Trolox equivalents (TE) g−1 indicating that the buriti oil comprises a reliable source of antioxidant compounds that can be more explored for food purposes.

DgCr22.1b, um isolado endofítico de Talaromyces sp., foi coletado na Floresta Amazônica a partir da planta medicinal Duguetia stelechantha. Do fracionamento do extrato metanólico de sua massa micelial, foi isolada uma nova xantona dimérica talaroxantona. A estrutura deste novo composto foi elucidada com base em análises espectroscópicas incluindo experimentos de ressonância magnética nuclear (RMN) 2D.

DgCr22.1b, an endophytic isolate of Talaromyces sp., was collected in the Amazonian Rainforest from the medicinal plant Duguetia stelechantha. From the fractionation of the methanolic mycelial extract, a new xanthone dimer talaroxanthone was isolated. The structure of this new compound was elucidated based on spectroscopic analyses including 2D nuclear magnetic resonance (NMR) experiments.

In vitro evaluation of alkaloidal fractions of twigs, barks and leaves from two Unonopsis species, Unonopsis guatterioides R.E. Fr. and Unonopsis duckei R.E. Fr., Annonaceae, against promastigote forms of Leishmania amazonensis revealed these species as sources of substances with promising leishmanicidal potential. All alkaloidal fractions from twigs, barks and leaves of U. guatterioides were classified as highly active, with IC50 1.07, 1.90, and 2.79 mg/mL, respectively. Only the alkaloidal fraction from the twigs of U. duckei was classified as inactive.

Por meio de técnicas cromatográficas, um novo peptídeo aniônico, denominado otacidina, foi isolado das secreções cutâneas de três anuros brasileiros Osteocephalus taurinus. O sequenciamento de novo dos aminoácidos foi realizado por meio de análises por espectrometria de massas por tempo de vôo acoplada à ionização dessortiva de matriz por laser (MALDI-TOF-MS/MS). O potencial antimicrobiano deste novo metabólito foi avaliado contra cepas de Escherichia coli e Pseudomonas aeruginosa, sendo observadas concentrações inibitórias mínimas (MICs) de 37,4 e 78,4 mM, respectivamente. Este trabalho é apenas o segundo relato de um peptídeo aniônico com propriedades antimicrobianas, isolado da pele de um anfíbio.

By means of chromatographic techniques, a novel anionic peptide, named otacidin, was isolated from the skin secretions of tree Brazilian anurans Osteocephalus taurinus. The de novo sequencing of the amino acids was performed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS) analyses. The antimicrobial potential of this novel metabolite was evaluated against Escherichia coli and Pseudomonas aeruginosa, being observed minimum inhibitory concentration (MICs) of 37.4 and 78.4 mM, respectively. This work is just the second report of an antimicrobial anionic peptide from an amphibian.

Mauritia flexuosa L. f., Arecaceae, is an endemic species of South America. This species was studied with the intent to isolate the constituents of its roots. After the fractionation of the n-hexane and methanolic extracts from the roots of M. flexuosa, six triterpenes were obtained: friedelin, taraxerone, lupenyl acetate, lupenone, betulin and betulinic acid, along with three flavonoids: rutin, quercitrin and quercetin. All the compounds were identified by analysis of NMR and MS data and comparison with the literature. All those compounds are been reported for the first time in Mauritia, and the chemosystematic significance of the flavonoids isolated in this genus is discussed.