Improving annotation propagation on molecular networks through random walks: introducing ChemWalker.

MOTIVATION: Annotation of the mass signals is still the biggest bottleneck for the untargeted mass spectrometry analysis of complex mixtures. Molecular networks are being increasingly adopted by the mass spectrometry community as a tool to annotate large-scale experiments. We have previously shown that the process of propagating annotations from spectral library matches on molecular networks can be automated using Network Annotation Propagation (NAP). One of the limitations of NAP is that the information for the spectral matches is only propagated locally, to the first neighbor of a spectral match. Here, we show that annotation propagation can be expanded to nodes not directly connected to spectral matches using random walks on graphs, introducing the ChemWalker python library. RESULTS: Similarly to NAP, ChemWalker relies on combinatorial in silico fragmentation results, performed by MetFrag, searching biologically relevant databases. Departing from the combination of a spectral network and the structural similarity among candidate structures, we have used MetFusion Scoring function to create a weight function, producing a weighted graph. This graph was subsequently used by the random walk to calculate the probability of 'walking' through a set of candidates, departing from seed nodes (represented by spectral library matches). This approach allowed the information propagation to nodes not directly connected to the spectral library match. Compared with NAP, ChemWalker has a series of improvements, on running time, scalability and maintainability and is available as a standalone python package. AVAILABILITY AND IMPLEMENTATION: ChemWalker is freely available at https://github.com/computational-chemical-biology/ChemWalker. CONTACT: ridasilva@usp.br. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Characterization of the fragmentation mechanisms in electrospray ionization tandem mass spectrometry of chloroquinoline derivatives with larvicidal activity against Aedes aegypti.

RATIONALE: 4,7-Dichloroquinoline (DCQ) represents a group of synthetic molecules inspired by natural products with important roles in biological and biomedical areas. This work aimed to characterize DCQ and its derivatives by high-resolution electrospray ionization (ESI) mass spectrometry and tandem mass spectrometry (ESI-MS/MS), supported by theoretical calculations. Biological assays were carried out with DCQ and its derivatives to determine LC50 values against Aedes aegypti larvae. METHODS: Five DCQ derivatives were synthesized by using previously described protocols. ESI-MS/MS analyses were carried out with a quadrupole/time-of-flight and ion-trap instrument. The proposed gas-phase protonation sites and fragmentation were supported by density functional theory calculations. The larvicidal tests were performed with the Ae. aegypti Rockefeller strain, and the LC50 values were determined by employing five test concentrations. Larval mortality was determined after treatment for 48 h. RESULTS: DCQ bromides or aldehydes (C-3 or C-8 positions), as well as the trimethylsilyl derivative (C-3 position), were prepared. Detailed ESI-MS/MS data revealed heteroatom elimination through an exception to the even-electron rule, to originate open-shell species. Computational studies were used to define the protonation sites and fragmentation pathways. High activity of DCQ and its derivatives against Ae. aegypti larvae was demonstrated. CONCLUSION: Our results provided a well-founded characterization of the fragmentation reactions of DCQ and its derivatives, which can be useful for complementary studies of the development of a larvicidal product against Ae. aegypti.

FTY720 increases paclitaxel efficacy in cisplatin-resistant oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma has high recurrence and cisplatin resistance. As cancer stem cells, autophagy, and sphingolipids have been appointed as associated with chemotherapy resistance, we tested combined treatments targeting autophagy and/or sphingolipid metabolism with paclitaxel using cisplatin-resistant oral squamous cell carcinoma cells. METHODS: Cisplatin-resistant oral squamous cell carcinoma cells were maintained under exposition to FTY720 and chloroquine combined with paclitaxel and submitted to viability, clonogenicity, and spheres formation assays. The xenograft tumor model using cisplatin-resistant CAL27 cells was adopted to examine the drug combinations' potential antitumoral efficacy. Using an animal model, sphingolipids profiles from plasma and tissue samples were obtained by liquid chromatography coupled to mass spectrometry to identify potential lipids associated with drug response. RESULTS AND DISCUSSION: Our results showed higher autophagic flux in cisplatin-resistant Ooral squamous cell carcinoma (CAL27 and SCC9) cells than in parental cells. The combinations of an autophagy inhibitor (chloroquine) or an autophagy inducer/sphingosine kinase 1 antagonist (FTY720) with paclitaxel (PTX) had a synergistic antitumor effect. Treated CisR cells lost clonogenicity and tumor sphere abilities and reduced proteins associated with proliferation, survival, and cancer stem cells. FTY720 plus PTX had higher antitumor efficacy than PTX against CAL27 CisR xenograft tumor formation. Additionally, increases in glucosylceramide, dehydroglucosylceramide, and sphingomyelin were presented in responsive tumors. CONCLUSION: FTY720 sensitizes cisplatin-resistant oral squamous cell carcinoma cells for paclitaxel.

A Comprehensive Review of Patented Antimicrobial Peptides from Amphibian Anurans.

Since the 1980s, studies of antimicrobial peptides (AMPs) derived from anuran skin secretions have unveiled remarkable structural diversity and a wide range of activities. This study explores the potential of these peptides for drug development by examining granted patents, amino acid modifications related to patented peptides, and recent amphibians' taxonomic updates influencing AMP names. A total of 188 granted patents related to different anuran peptides were found, with Asia and North America being the predominant regions, contributing 65.4% and 15.4%, respectively. Conversely, although the Neotropical region is the world's most diversified region for amphibians, it holds only 3.7% of the identified patents. The antimicrobial activities of the peptides are claimed in 118 of these 188 patents. Additionally, for 160 of these peptides, 66 patents were registered for the natural sequence, 69 for both natural and derivative sequences, and 20 exclusively for sequence derivatives. Notably, common modifications include alterations in the side chains of amino acids and modifications to the peptides' N- and C-termini. This review underscores the biomedical potential of anuran-derived AMPs, emphasizing the need to bridge the gap between AMP description and practical drug development while highlighting the urgency of biodiversity conservation to facilitate biomedical discoveries.

Influence of Host Plants and Tending Ants on the Cuticular Hydrocarbon Profile of a Generalist Myrmecophilous Caterpillar.

In myrmecophilous organisms, which live in symbiosis with ants, cuticular hydrocarbons (CHCs) play a pivotal role in interspecific communication and defense against chemical-oriented predators. Although these interactions form complex information webs, little is known about the influence of biotic environmental factors on the CHC profiles of myrmecophiles. Here, we analyzed the effect of different host plants and tending ants on the larval CHC profile of Synargis calyce (Lepidoptera: Riodinidae), a polyphagous species with facultative myrmecophily. Groups of caterpillars were fed individually with three host plant species (without tending ants), and with two tending ant species. Through gas chromatography analysis, we compared the cuticular profiles of treatments and found a high similarity between plants and caterpillars (65-82%), but a low similarity between caterpillars and their tending ants (30 - 25%). Cluster analysis showed that caterpillars, ants, and plants form distinct groups, indicating that S. calyce caterpillars have their own chemical profile. These results are similar to those observed for Lycaenidae caterpillars indicating that there is functional convergence in the chemical strategies used by myrmecophilous caterpillar species with similar ecology. Also, the results suggest that the cuticular compounds of S. calyce are primarily influenced by their host plants rather than their tending ants. Thus, we propose that these caterpillars present a trade-off between camouflage and directly informing their presence to ants, maintaining their unique chemical profile, though slightly affected by biotic environmental factors.

Antinociceptive Effect of Dillenia indica (Linn.) Mediated by Opioid and Cannabinoid Systems: Pharmacological and Chemical Studies.

Dillenia indica (Linn.) has been reported by several biological activities, including anti-inflammatory, antioxidant, antidiabetic, anti-hyperglycemic, antiproliferative, antimutagenic, anticholinesterase, and antimicrobial. In Brazilian traditional medicine, the fruits of D. indica have been used to treat general topical pain and inflammation, but with no scientific validation. Thus, aiming to study its chemical constitution and antinociceptive properties, the crude extract (CE) and fractions obtained from the fruits of D. indica were submitted to an in vivo pharmacological evaluation and a dereplication study by LC-MS/MS analysis, assisted by the Global Natural Product Social Molecular Networking (GNPS). The oral antinociceptive activity of the fruits of D. indica and the possible participation of the opioid and cannabinoid systems were demonstrated in the formalin-induced nociception model. The chemical dereplication study led us to identify several known chemical constituents, including flavonoids, such as caffeoylmalic acid, naringenin, quercetin, and kaempferol. According to literature data, our results are compatible with significant antinociceptive and anti-inflammatory activities. Therefore, the flavonoid constituents of the fruits of D. indica are probably responsible for its antioxidant, anti-inflammatory, and antinociceptive effects mediated by both opioid and cannabinoid systems, confirming its folk use in the treatment and relief of pain.

Unlocking Hidden Treasures: LC-MS/MS Molecular Networks for Exploring Novel Passiflora Species with Pharmaceutical Potential.

In this study, we report the metabolic profiling of several previously uncharacterized Passiflora species native to Brazil, employing molecular networks to delve deeper into chemical constituents. Using the GNPS platform, in silico tools, and substructure annotation techniques, we expanded the chemical annotations. Principal Coordinate Analysis (PCoA) revealed significant metabolic similarities between several species, including P. incarnata, suggesting shared pharmacological potential. Our identification of metabolic compounds facilitated comparisons between understudied species with medicinal properties. Notably, we documented 25 previously uncharacterized species, paving the way for the development of novel products aimed at improving human well-being. This research focused on several native Passiflora species from Brazil, highlighting their unexplored therapeutic potential.

Photoprotective Potential of Passiflora Cincinnata Mast. (Passifloraceae) Hydro-Alcoholic Extracts.

Passiflora cincinnata is a Passifloraceae typical of the Caatinga, a biome unique to Brazil. It has various pharmacological properties associated with its high flavonoid content. Vitexin, isovitexin, orientin, isoorientin and derivatives are the main chemical and pharmacological markers for this plant. Although flavonoids enriched-extracts have been widely applied in phytocosmetics, especially in sunscreen formulations, the use of P. cincinnata as a photoprotective ingredient remains unexplored. Different hydro-alcoholic extracts were prepared and their antioxidant and photoprotective activities were evaluated by in vitro assays. The most promising extract (Pc-1) was analyzed by HPLC-DAD-ESI-MS/MS. Nine flavonoids were identified as major compounds: isovitexin-7-O-glucoside, isoorientin-2"O-hexoside, orientin, isoorientin, isovitexin-2"-O-glucoside, isovitexin-6"-O-glucoside, isoscoparin and isoquercitrin. Finally, Pc-1 (5 and 10 %, v/v) was incorporated into gel formulations, alone or combined to commercial chemical filters (benzophenone-3 and octyl methoxycinnamate). Formulations containing Pc-1 showed high SPFspectrophotometric values. When combined to commercial filters, Pc-1 (5 %) potentiated their photoprotective efficacy (p<0.05). A physicochemical characterization indicated no incompatibility or signs of instability after extract incorporation. Altogether, these findings encourage the use of Pc-1 as a photoprotective ingredient or co-adjuvant in sunscreens formulations.

Are benzoylium and nitrilium ions produced from substituted 2-arene-2-oxazolines during mass spectrometry? A study based on density functional theory calculations, quantum theory of atoms in molecules, and electrospray ionization tandem mass spectrometry.

RATIONALE: Oxazolines are important compounds for drug development, synthesis, and pharmaceutical applications. Interest in analyzing and developing methods to characterize reaction products from these small heterocyclics has led us to study the gas-phase reactivity and fragmentation of seven 2-arene-2-oxazolines compounds using computational chemistry combined with mass spectrometry. METHOD: Protonation sites were investigated using computed proton affinity, gas-phase basicity, and some quantum chemistry descriptors of reactivity; the B3LYP/6-31+G(d,p) computational model was used. Fragmentation mechanisms were suggested by employing data from collision-induced dissociation (CID), energy-resolved plots from MS/MS spectra, multiple-stage experiments, and survival-yield method. RESULTS: Protonation studies based on quantum theory of atoms in molecules (QTAIM) and computational thermochemistry were useful to describe the reactivity of the investigated 2-arene-2-oxazolines, which can be protonated at the nitrogen atom. Three major fragmentation pathways were identified for the protonated molecules: formation of (a) benzoylium or (b) nitrilium ions through elimination of 71 and 72 u from the protonated molecules, respectively, and (c) elimination of 54 u from [M+H]+ . These pathways were exploited by the density functional theory calculations combined with QTAIM studies. CONCLUSIONS: Our results can help in identifying 2-arene-2-oxazoline derivatives using electrospray ionization tandem mass spectrometry (ESI-MS/MS), which can be applied for monitoring reactions through the identified diagnostic ions (product ions). Also, we can suggest that benzoylium and nitrilium ions emerge during fragmentation under CID conditions.

Oxidation Products from the Neolignan Licarin A by Biomimetic Reactions and Assessment of in vivo Acute Toxicity.

Licarin A, a dihydrobenzofuranic neolignan presents in several medicinal plants and seeds of nutmeg, exhibits strong activity against protozoans responsible for Chagas disease and leishmaniasis. From biomimetic reactions by metalloporphyrin and Jacobsen catalysts, seven products were determined: four isomeric products yielded by epoxidation from licarin A, besides a new product yielded by a vicinal diol, a benzylic aldehyde, and an unsaturated aldehyde in the structure of the licarin A. The incubation with rat and human liver microsomes partially reproduced the biomimetic reactions by the production of the same epoxidized product of m/z 343 [M + H]+. In vivo acute toxicity assays of licarin A suggested liver toxicity based on biomarker enzymatic changes. However, microscopic analysis of tissues sections did not show any tissue damage as indicative of toxicity after 14 days of exposure. New metabolic pathways of the licarin A were identified after in vitro biomimetic oxidation reaction and in vitro metabolism by rat or human liver microsomes.

Neuroprotective Properties of Chlorogenic Acid and 4,5-Caffeoylquinic Acid from Brazilian arnica (Lychnophora ericoides) after Acute Retinal Ischemia.

Lychnophora is a genus of South American flowering plants in the daisy family, popularly known as "Brazilian arnica". It is used in traditional medicine as an anti-inflammatory and analgesic agent, whose active components are derived from chlorogenic acid (CGA) and C-flavonoids. Since the drugs currently used are ineffective to treat glaucoma, agents with antioxidant and anti-inflammatory properties may represent new alternatives in preventing cellular lesions in retinal ischemia. In this study, we report the neuroprotective effects of CGA and 4,5-di-O-[E]-caffeoylquinic (CQA) acid, isolated from Lychnophora plants, in a rodent glaucoma model. Wistar rats were administered intravitreally with 10 µg CGA or CGA, and then subjected to acute retinal ischemia (ISC) by increasing intraocular pressure (IPO) for 45 minutes followed (or not) by 15 minutes of reperfusion (I/R). Qualitative and quantitative analyses of neurodegeneration were performed using hematoxylin-eosin or Fluoro-Jade C staining protocols. All retinas submitted to ISC or I/R exhibited matrix disorganization, pyknotic nuclei, and pronounced vacuolization of the cytoplasm in the ganglion cell layer (GCL) and inner nuclear layer (INL). Pretreatment with CGA or CQA resulted in the protection of the retinal layers against matrix disorganization and a reduction in the number of vacuolized cells and pyknotic nuclei. Also, pretreatment with CGA or CQA resulted in a significant reduction in neuronal death in the GCL, the INL, and the outer nuclear layer (ONL) after ischemic insult. Our study demonstrated that CGA and CQA exhibit neuroprotective activities in retinas subjected to ISC and I/R induced by IPO in Wistar rats.

Cytotoxic Cyclotides from Anchietea pyrifolia, a South American Plant Species.

Cyclotides are mini-proteins with potent bioactivities and outstanding potential for agricultural and pharmaceutical applications. More than 450 different plant cyclotides have been isolated from six angiosperm families. In Brazil, studies involving this class of natural products are still scarce, despite its rich floristic diversity. Herein were investigated the cyclotides from Anchietea pyrifolia roots, a South American medicinal plant from the family Violaceae. Fourteen putative cyclotides were annotated by LC-MS. Among these, three new bracelet cyclotides, anpy A-C, and the known cycloviolacins O4 (cyO4) and O17 (cyO17) were sequenced through a combination of chemical and enzymatic reactions followed by MALDI-MS/MS analysis. Their cytotoxic activity was evaluated by a cytotoxicity assay against three human cancer cell lines (colorectal carcinoma cells: HCT 116 and HCT 116 TP53-/- and breast adenocarcinoma, MCF 7). For all assays, the IC50 values of isolated compounds ranged between 0.8 and 7.3 µM. CyO17 was the most potent cyclotide for the colorectal cancer cell lines (IC50, 0.8 and 1.2 µM). Furthermore, the hemolytic activity of anpy A and B, cyO4, and cyO17 was assessed, and the cycloviolacins were the least hemolytic (HD50 > 156 µM). This work sheds light on the cytotoxic effects of the anpy cyclotides against cancer cells. Moreover, this study expands the number of cyclotides obtained to date from Brazilian plant biodiversity and adds one more genus containing these molecules to the list of the Violaceae family.

Combination of GC-MS Molecular Networking and Larvicidal Effect against Aedes aegypti for the Discovery of Bioactive Substances in Commercial Essential Oils.

Dengue is a neglected disease, present mainly in tropical countries, with more than 5.2 million cases reported in 2019. Vector control remains the most effective protective measure against dengue and other arboviruses. Synthetic insecticides based on organophosphates, pyrethroids, carbamates, neonicotinoids and oxadiazines are unattractive due to their high degree of toxicity to humans, animals and the environment. Conversely, natural-product-based larvicides/insecticides, such as essential oils, present high efficiency, low environmental toxicity and can be easily scaled up for industrial processes. However, essential oils are highly complex and require modern analytical and computational approaches to streamline the identification of bioactive substances. This study combined the GC-MS spectral similarity network approach with larvicidal assays as a new strategy for the discovery of potential bioactive substances in complex biological samples, enabling the systematic and simultaneous annotation of substances in 20 essential oils through LC50 larvicidal assays. This strategy allowed rapid intuitive discovery of distribution patterns between families and metabolic classes in clusters, and the prediction of larvicidal properties of acyclic monoterpene derivatives, including citral, neral, citronellal and citronellol, and their acetate forms (LC50 < 50 µg/mL).

Time-Scale Shifting of Volatile Semiochemical Levels in Wild Type Lychnophora ericoides (Brazilian arnica) and Pollinator Records.

Lychnophora ericoides is a Brazilian folk phytomedicine from Cerrado's "campus rupestris". Its volatile organic compounds includes bisabolene-derivatives as major compounds. Herein we provide the chemical profiling of constitutive volatile sesquiterpenes from L. ericoides leaves, timeframe emissions surveys, and pollinators records. In situ samples of L. ericoides were harvested. A headspace-solid phase micro extraction method of pre-concentration was optimized. Identification was done through GC-MS. Isolation and structural elucidation were performed whenever necessary. Pollinators were registered in pictures and video. Short time-series and harmonic regressions determined rhythms of single compounds, and average chromatographic signal area was used to determine mono and sesquiterpene rhythms. Concluding, optimized headspace-solid phase micro extraction method of terpenes level analysis was reached. α-Pinene, ß-pinene, α-terpinene, para-cymene, limonene, γ-terpinene, terpinen-4-ol, dehydro-sesquicineole, and ß-guaiene were identified using GC-MS data. 11-dehydro cadinol and ortho-acetoxy bisabolol were elucidated. Sesquiterpenes concentrations were higher due to temperature rise, lower leaf age, and flowering seasons. Harmonic regressions determined that daylight might control levels of terpenes. Hummingbird, hemiptera insects, and wasps were recorded visiting Compositae capitulum for the first time. We studied nondomestic plants from in situ conditions and concluded that bisabolene-derivative levels were more abundant than monoterpenes during flowering throughout the summer.

Anticancer Potential of Compounds from the Brazilian Blue Amazon.

"Blue Amazon" is used to designate the Brazilian Economic Exclusive Zone, which covers an area comparable in size to that of its green counterpart. Indeed, Brazil flaunts a coastline spanning 8000 km through tropical and temperate regions and hosting part of the organisms accredited for the country's megadiversity status. Still, biodiversity may be expressed at different scales of organization; besides species inventory, genetic characteristics of living beings and metabolic expression of their genes meet some of these other layers. These metabolites produced by terrestrial creatures traditionally and lately added to by those from marine organisms are recognized for their pharmaceutical value, since over 50% of small molecule-based medicines are related to natural products. Nonetheless, Brazil gives a modest contribution to the field of pharmacology and even less when considering marine pharmacology, which still lacks comprehensive in-depth assessments toward the bioactivity of marine compounds so far. Therefore, this review examined the last 40 years of Brazilian natural products research, focusing on molecules that evidenced anticancer potential-which represents ~ 15% of marine natural products isolated from Brazilian species. This review discusses the most promising compounds isolated from sponges, cnidarians, ascidians, and microbes in terms of their molecular targets and mechanisms of action. Wrapping up, the review delivers an outlook on the challenges that stand against developing groundbreaking natural products research in Brazil and on a means of surpassing these matters.

Engineering of galectin-3 for glycan-binding optical imaging.

Galectin-3 (Gal-3) is a multifunctional glycan-binding protein that participates in many pathophysiological events and has been described as a biomarker and potential therapeutic target for severe disorders, such as cancer. Several probes for Gal-3 or its ligands have been developed, however both the pathophysiological mechanisms and potential biomedical applications of Gal-3 remain not fully assessed. Molecular imaging using bioluminescent probes provides great sensitivity for in vivo and in vitro analysis for both cellular and whole multicellular organism tracking and target detection. Here, we engineered a chimeric molecule consisting of Renilla luciferase fused with mouse Gal-3 (RLuc-mGal-3). RLuc-mGal-3 preparation was highly homogenous, soluble, active, and has molecular mass of 65,870.95 Da. This molecule was able to bind to MKN45 cell surface, property which was inhibited by the reduction of Gal-3 ligands on the cell surface by the overexpression of ST6GalNAc-I. In order to obtain an efficient and stable delivery system, RLuc-mGal-3 was adsorbed to poly-lactic acid nanoparticles, which increased binding to MKN45 cells in vitro. Furthermore, bioluminescence imaging showed that RLuc-mGal-3 was able to indicate the presence of implanted tumor in mice, event drastically inhibited by the presence of lactose. This novel bioluminescent chimeric molecule offers a safe and highly sensitive alternative to fluorescent and radiolabeled probes with potential application in biomedical research for a better understanding of the distribution and fate of Gal-3 and its ligands in vitro and in vivo.

Characterization of aporphine alkaloids by electrospray ionization tandem mass spectrometry and density functional theory calculations.

RATIONALE: Aporphine alkaloids represent a large group of isoquinoline natural products with important roles in biological and biomedical areas. Their characterization by electrospray ionization tandem mass spectrometry (ESI-MS/MS) can contribute to their rapid identification in complex biological matrices. METHODS: We report the fragmentation of protonated 7,7-dimethylaporphine alkaloids by ESI-MS/MS, and the putative annotation of aporphine alkaloids in plant extracts. We used low- and high-resolution MS/MS analyses to rationalize the fragmentation pathways, and employed the B3LYP/6-31 + G(d,p) density functional theory (DFT) model to provide thermochemical parameters and to obtain the reactive sites. RESULTS: DFT calculations of a set of 7,7-dimethylaporphine alkaloids suggested the heterocyclic amino group as the most basic site due to the proton affinity of the nitrogen atom. Collision-induced dissociation experiments promoted • OCH3 elimination instead of the expected neutral loss of the heterocyclic amino group, pointing to the [M - 15 + H]•+ ion as the diagnostic fragment for 7,7-dimethylaporphine alkaloids. The analysis of plant extracts led to the annotation of 25 aporphine alkaloids. Their fragmentation initiated with the loss of the amino group followed by formation of a cyclic carbocation. Further reactions derived from consecutive charge-remote and/or charge-induced fragmentations of the substituents attached to the aromatic system. The mechanisms were re-examined based on plausible gas-phase ion chemistry reactions. CONCLUSIONS: Taken together, the diagnostic product ions and the series of radical and neutral eliminations provided information about the location of methylenedioxy, aromatic methoxy, and vicinal methoxy and hydroxy groups in aporphine alkaloids, assisting their characterization via MS/MS.

Characterization of 3-aminospirostane alkaloids from roots of Solanum paniculatum L. with hepatoprotective activity.

RATIONALE: Solanum paniculatum L., popularly known as jurubeba, has traditionally been used in Brazilian folk medicine for liver diseases. However, there is a lack of knowledge about the chemical characterization of 3-aminospirostane alkaloids, an important class related to pharmacological activities. This work aimed to characterize the alkaloids using liquid chromatography with tandem mass spectrometry (LC/MS/MS) supported by molecular networking and theoretical calculations as well as to evaluate the contribution to hepatoprotective activity. METHODS: S. paniculatum roots were collected and macerated with MeOH/H2 O (8:2) obtaining the crude extract (SP-CE). From this, partition using EtOAc with pH variation yielded the alkaloidic fraction (SP-AF). Both were evaluated in an acute liver injury model (100 and 200 mg/kg), after intraperitoneal administration of carbon tetrachloride (CCl4 ) in mice. AST (aspartate transaminase) and ALT (alanine transaminase) serum levels were investigated, as well as the histopathological characteristics. The SP-CE and SP-AF were analyzed by LC/MS/MS, using quadrupole/time-of-flight and ion-trap systems. The alkaloids annotated by the GNPS molecular network had their structures defined using gas-phase ionization and fragmentation reaction supported by theoretical calculations. RESULTS: The SP-CE and SP-AF decreased the ALT serum levels compared with the negative control. The group treated with the SP-CE (at the highest dose) demonstrated a significant decrease of ALT. Hepatic cell degeneration decrease was observed mainly at the highest dose of the treatment. Detailed electrospray ionization MS/MS data allowed us to identify alkaloids not previously reported, to propose their gas-phase reactions and to redefine the initial open ring fragmentation mechanism of the steroidal alkaloids with the jurubidine moiety. CONCLUSIONS: The results allowed us to identify seven steroidal alkaloids from jurubeba and redefine the initial mechanism of fragmentation. A significant hepatoprotective effect was also demonstrated, corroborating its traditional use.

Fragmentation study of clerodane diterpenes from Casearia species by tandem mass spectrometry (quadrupole time-of-flight and ion trap).

RATIONALE: Clerodane-type diterpenes from Casearia species show important pharmacological activites such as antitumor, antimicrobial and anti-inflamatory. There are several mass spectrometry (MS)-based methods for identification of diterpenes; however, there is still a lack of MS procedures capable of providing characteristic fragmentation pathways for a rapid and unambiguous elucidation of casearin-like compounds. METHODS: Casearin-like compounds were investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The fragmentation studies were carried out by tandem mass spectrometry in space (quadrupole time-of-flight (QTOF)) using different collision energies and also by tandem mass spectrometry in time (QIT) by selective isolation of product ions. RESULTS: Casearin-like compounds presented a predominance of sodium- and potassium-cationized precursor ions. Both QIT and QTOF techniques provided sequential neutral losses of esters related to the R1 to R5 substituents linked to the nucleus of the clerodane diterpenes. The fragmentation pathway is initiated with a cleavage of the ester moieties R2 followed by the elimination of the ester groups R3 , both losing neutral carboxylic acids. Using QIT, it was also possible to observe the cleavage of the ester groups R1 or R5 by MS4 experiments. CONCLUSIONS: Through a rational analysis of the fragmentation mechanisms of Casearia diterpenes it was possible to suggest an annotation strategy based on the sequential cleavages of the ester groups related to the R2 , R3 and R5 substituents. These results will assist studies of the dereplication and metabolomics involving casearin-like compounds present in complex extracts of Casearia species.

Rhodnius spp. are differentiated based on the peptide/protein profile by matrix-assisted laser desorption/ionization mass spectrometry and chemometric tools.

Triatominae are hematophagous insects involved in the transmission of Chagas disease. Among the 19 genera of the subfamily, those with the highest epidemiological importance regarding the dissemination of Trypanosoma cruzi are Panstrongylus, Rhodnius, and Triatoma. Of these three genera, Rhodnius presents the greatest difficulties for specific identification. Thus, there is a need to overcome the difficulties in identifying phenotypes of similar species of this genus. In the present study, the MALDI-TOF MS methodology was used to identify 12 Rhodnius species, among the 21 admitted. The MALDI-TOF MS methodology allowed specific characterization through the identification of peptides and proteins, starting from four different methods of extraction: (A) acetonitrile/formic acid (ACN/AF), (B) acetonitrile/trifluoroacetic acid (ACN/TFA), (C) isopropyl/formic acid (IPA/AF), and (D) methanol/formic acid (MeOH/AF), and four types of MALDI-TOF matrices: α-cyano-4-hydroxycinnamic acid (CHCA), sinapic acid (SA), 6-aza-2-thiothymine (ATT), and 2,6-dihydroxyacetophenone (DHAP). The experiments were performed by combining the four solvents and four matrices to select the best MALDI extraction/matrix. The application of the MALDI-TOF MS technique, through the digital mass spectrometry approach combined with chemometric tools, such as partial least squares-discriminant analysis (PLS-DA), was able to discriminate 12 species of Rhodnius genus, which are difficult to identify using morphological characteristics. Thus, in view of the results obtained, the methodology described in the present article can be applied with speed and efficiency for the discrimination of Triatominae species. Graphical Abstract.