Search

The Amazon rainforest is renowned for its biodiversity and as a reservoir of edible and medicinal plants. The phytochemicals in murici and taperebá fruits serve as natural antioxidants, contributing to cultural preservation, ecosystem protection, and economic opportunities. However, limited scientific research on their composition and health benefits hinders their recognition as functional foods. This study aimed to evaluate the antioxidant activity, carotenoid content, phenolic compounds, and antitumor effects of murici and taperebá fruit pulps. Four antioxidant tests (2,2-Diphenyl-1-picrylhydrazylradical scavenging activity, 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) method, 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) method, oxygen radical absorbance capacity) were conducted, and total phenolics were quantified (Folin-Ciocalteu). Phenolics were identified using UHPLC-HRMS, and carotenoids by high-performance liquid chromatography (HPLC). The impact on breast cancer cell viability (MCF-7, MDA-MB-231) was assessed via water-soluble tetrazolium (WST) assay. Both fruits showed high antioxidant activity and phenolic content, with murici leading. HPLC revealed five carotenoids per fruit, with taperebá showing higher concentrations. UHPLC-HRMS identified 23 phenolic compounds: 16 in murici aqueous extract, 18 in murici ethanolic extract, and 15 in each taperebá extract. WST assay demonstrated that both fruits exerted a significant impact on breast cancer cells, reducing their viability in a dose-dependent manner. These findings underscore the potential of murici and taperebá as sources of phytochemical antioxidants and antiproliferative agents with promising health applications., (© 2025 Deutsche Pharmazeutische Gesellschaft.)

Coffee is one of the most important beverages in the world and is produced from Coffea spp. beans. Diterpenes with ent -kaurane backbones have been described in this genus, and substances such as cafestol and kahweol have been widely investigated, along with their derivatives and biological properties. Other coffee ent -kaurane diterpenoids have been reported with new perspectives on their biological activities. The aim of this review is to update the chemical diversity of ent -kaurane diterpenoids in green and roasted coffee, detailing each new compound and reporting its biological potential. A systematic review was performed using the bibliographic databases (SciFinder, Web of Science, ScienceDirect) and specific keywords such as "coffea diterpenes", "coffee diterpenes", "coffee ent-kaurane diterpenes" and "coffee diterpenoids". Only articles related to the isolation of coffee ent -kaurane compounds were considered. A total of 146 compounds were related to Coffea spp. since the first report in 1932. Different chemical skeletons were observed, and these compounds were grouped as furan-type, oxidation-type, rearrangement-type, lacton-type, and lactam-type, among others. In general, the new coffee diterpenoids showed potential as antidiabetic, antidiapogenic, α-glucosidade inhibition, antiplatelet activity, and Cav.3 inhibitors agents, revealing the possibilities for the design, discovery, and development of new drugs.

Background: The occurrence of co-infections during schistosomiasis, a neglected tropical disease, with other parasites have been reported suggesting an impaired host immune defense. Macrophage purinergic P2X7 receptor (P2X7R) plays an important role against intracellular pathogens. Therefore, we investigated the P2X7R-mediated phagocytosis and killing capacity of Leishmania amazonensis by macrophages during schistosomiasis in vitro and in vivo., Methods: Swiss and C57BL/6 (Wild type) and P2X7R -/- were randomized in two groups: control (uninfected) and Schistosoma mansoni-infected. Alternatively, control Swiss and S. mansoni-infected mice were also infected with L. amazonensis., Results: The pre-treatment of control macrophages with the P2X7R antagonist (A74003) or TGF-β reduced the phagocytosis index, mimicking the phenotype of cells from S. mansoni-infected mice and P2X7R -/- mice. Apyrase also reduced the phagocytosis index in the control group corroborating the role of ATP to macrophage activation. Moreover, l-arginine-nitric oxide pathway was compromised during schistosomiasis, which could explain the reduced killing capacity in response to ATP in vitro and in vivo. We found an increased extracellular nucleotide (ATP, ADP and AMP) hydrolysis along with an increased frequency of F4/80 + CD39 + macrophages from the S. mansoni-infected group. Moreover, the content of adenosine in the cell supernatant was higher in the S. mansoni-infected group in relation to controls. Schistosomiasis also increased the expression of macrophage adenosine A 2B R. In good accordance, both ADA and the selective A 2B R antagonist restored the phagocytosis index of macrophages from S. mansoni-infected group., Conclusions: Altogether, the altered P2X7R and A 2B R signaling limits the role of macrophages to host defense against L. amazonensis during schistosomiasis, potentially contributing to the pathophysiology and clinically relevant co-infections., Competing Interests: Declaration of competing interest none., (© 2024 The Authors. Published by Elsevier B.V. on behalf of Chang Gung University. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Trema micranthum (Cannabaceae) has emerged as a promising new source of cannabinoids, including cannabidiol (CBD). Given the substantial medicinal demand for cannabinoids and the regulatory challenges associated with Cannabis sativa due to the presence of Δ 9 -tetrahydrocannabinol (THC), this study sought to explore the presence of CBD, THC, and their precursors, Δ 9 -tetrahydrocannabinolic acid A (THCA A) and cannabidiolic acid (CBDA), in various parts of Trema micranthum using UHPLC-HRMS/MS (Orbitrap). Extracts from fruits, leaves, inflorescences, and stems were obtained using a methanol/hexane (9:1, v/v) solvent mixture. UHPLC coupled with an Orbitrap mass spectrometer was employed for cannabinoid identification and quantification, with standard mixtures prepared in methanol. The extracts yielded significant quantities, such as 6.6%/g from leaves and 3%/g from fruits. Cannabinoids were detected in fruits, leaves, and inflorescences, with acidic forms (CBDA and THCA A) present in higher concentrations than their neutral counterparts. Notably, leaves contained 4.43 × 10 -3 µg/g of CBD and 1.05 ×  10 -3 µg/g of THC. These findings, facilitated by high-resolution analytical methods, underscore the potential of Trema micranthum as an alternative source for cannabinoids, guiding future research in this area., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

One of the principal byproducts of coffee roasting is the coffee parchment. It is abundant in bioactive substances, including derivatives of chlorogenic acids, which are well-known for their exceptional antioxidant effects. It is advantageous to use environmentally friendly extraction techniques on such residues since it adds value to the entire coffee production process supply chain. The aim of this work was to assess and enhance the ability of non-conventional extraction techniques to extract derivatives of chlorogenic acid from coffee parchment. A central composite design was used to maximize the recovery of those phenolic compounds. The optimized extraction conditions were with 5 min extraction period, at a temperature of 70 °C, and 80% ethanol in the extractor solvent. In this conditions extraction recovery of chlorogenic acids was of 0.8% by the use of microwave-aided extraction (MAE). The optimized conditions are practical, economical, and ecologically friendly method to extract phenolic compounds and, consequently, underscores the potential for sustainable utilization of coffee parchment, offering a valuable contribution to the development of environmentally conscious strategies within the coffee industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Lysergic acid diethylamide (LSD) is a synthetic psychedelic compound with potential therapeutic value for psychiatric disorders. This study aims to establish Caenorhabditis elegans as an in vivo model for examining LSD's effects on locomotor behavior. Our results demonstrate that LSD is absorbed by C. elegans and that the acute treatment reduces animal speed, similar to the role of endogenous serotonin. This response is mediated in part by the serotonergic receptors SER-1 and SER-4. Our findings highlight the potential of this nematode as a new experimental model in psychedelic research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: Maternal high-fat diet (HF) programs obesity, metabolic dysfunction-associated steatotic liver disease (MASLD), hypertriglyceridemia, and hyperglycemia associated with increased endocannabinoid system (ECS) in the liver of adult male rat offspring. We hypothesized that maternal HF would induce sex specific ECS changes in the liver of newborn rats, prior to obesity onset, and maternal fish oil (FO) supplementation would reprogram the ECS and lipid metabolism markers preventing liver triglycerides (TG) accumulation., Methods: Female rats received a control (CT) (10.9% fat) or HF (28.7% fat) diet 8 weeks prior to mating and during pregnancy. A subgroup of HF dams received 3% FO supplementation in the HF diet (35.4% fat) during pregnancy (HFFO). Serum hormones and liver TG, ECS, lipid metabolism, oxidative stress and autophagy markers were assessed in male and female newborn offspring., Results: Maternal HF diet increased liver cannabinoid receptor 1 (CB1) in males and decreased CB2 in females, with no effect on liver TG. Maternal FO supplementation reduced liver CB1 regardless of the offspring sex, but reduced TG liver content only in females. FO reduced the liver content of the endocannabinoid anandamide in males, and the content of 2-arachidonoylglycerol in both sexes. Maternal HF increased lipogenic and decreased lipid oxidation markers, and FO induced the opposite regulation in the liver of offspring., Conclusion: Prenatal HF and FO differentially modulate liver ECS in the offspring before obesity and MASLD development. These results suggest that maternal nutrition at critical stages of development can modulate the offspring's ECS, predisposing or preventing the onset of metabolic diseases., (© 2024. Springer-Verlag GmbH Germany, part of Springer Nature.)

Rotigotine (RTG) is a dopamine agonist used in the treatment of Parkinson's disease. As it is susceptible to oxidation, stability studies must be carefully designed for the identification and characterization of all possible degradation products. Here, RTG degradation was evaluated according to the International Conference on Harmonization guidelines under various stress conditions, including acidic and basic hydrolysis, oxidative, metallic, photolytic, and thermal conditions. Additionally, more severe stress conditions were applied to induce RTG degradation. Significant degradation was only observed under oxidative and photolytic conditions. The samples were analyzed by high performance liquid chromatography coupled to photodiode array detectors, charged aerosol, and high-resolution mass spectrometry. Chromatographic analyses revealed the presence of eight substances related to RTG, four of which were already described and were qualified impurities (impurities B, C, K and E) and four new degradation products (DP-1 - DP-4), whose structures were characterized by high-resolution mass spectrometry through Q-Orbitrap and electrospray ionization. In the stress testing of the active pharmaceutical ingredient in solid form, significant RTG degradation was observed in the presence of the oxidative matrix. The results corroborate the literature that confirm the high susceptibility of RTG to oxidation and the importance of using different detectors to detect degradation products in forced degradation studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Aqueous and hydroalcoholic extracts from the pulp of Ambelania acida Aubl. (Apocynaceae) fruits were subjected to analysis through UHPLC-HRMS and antioxidant potential using the TPC, DPPH, ABTS, FRAP, and ORAC assays. A putative identification of the compounds carried out by comparison of the fragmentation spectra revealed the predominance of the monoterpene indole alkaloids tabersonine, pseudocopsinine, ajmalicine, and strictosidine. Additionally, gallic acid, caffeic acid, citric acid, 3- O - p -coumaroylquinic acid, chlorogenic acid, catechin, ellagic acid, eschweilenol C (ellagic acid deoxyhexoside), and sucrose were identified. In face of the phenolic compounds observed, hydroalcoholic extract showed a higher antioxidant activity compared to the aqueous extract, observed at TPC (108.85 mg GAE/100g), FRAP (0.73 µmol Fe 2 SO 4 /g), DPPH (1221.76 µmol TE/g), ABTS (3460.00 µmol TE/g), and ORAC assays (120.47 µmol TE/g). These findings underscore the abundant presence of bioactive compounds, including phenolics and alkaloids, in an edible Amazonian fruit.

Ocotea , the largest genus in the Lauraceae family, encompasses numerous species of scientific interest. However, most Ocotea species have only been described morphologically. This study used an untargeted metabolomics workflow with UHPLC-HRMS and GNPS-FBMN to provide the first chemical evaluation of the polar specialized metabolites of O. delicata leaves. Leaves from three O. delicata specimens were extracted using ultrasound-assisted extraction with 70% ethanol. Among the examined samples, 44 metabolites, including alkaloids and flavonoids, were identified. In contrast to other Ocotea species, O. delicata has a wider diversity of kaempferol derivatives than quercetin. The biomass of the specimens showed a significant correlation with the chemical profile. The similarity among specimens was mostly determined by the concentrations of quinic acid, kaempferol glycosides, and boldine. The evaluated specimens exhibited chemical features similar to those of species classified as New World Ocotea , with the coexistence of aporphine and benzylisoquinoline alkaloids.

Dietary supplements (DS) are intended for healthy people to maintain or improve their overall health. Its consumption is widespread in large part of the general population and at all levels of athletes. Nevertheless, DS use can also pose health risks to individuals and, in the case of athletes, may lead to adverse analytical findings (AAFs) due to the possibility of DS contamination or adulteration with doping agents banned by the World Anti-Doping Agency. Although educational initiatives are being performed in Brazil to warn the sports community about inadvertent doping cases, AAFs connected to the DS administration have been increasingly growing. The findings of DS analyzed by the Brazilian Doping Control Laboratory (LBCD), between 2017 and 2022, after Testing Authorities (TAs) analysis requests, showed an alarming number of tainted samples. Diuretics were the most common adulterants found in all supplement types. However, the profile of prohibited substances in manufactured and compounded dietary supplements (MDS and CDS, respectively) were distinct, with stimulants being most prevalent in MDS and anabolic agents in CDS products. Additionally, MDS samples generally presented higher estimated concentrations of banned substances (mg/g) than CDS samples (μg/g). The common practice of DS intake by athletes continues to be of great concern for a doping-free sport, given the high prevalence of prohibited substances detected in the analyzed samples by the LBCD. The current Brazilian scenario reinforces the importance of raising awareness in the sports community of the possible consequences of an unintentional doping case linked to DS use., (© 2023 John Wiley & Sons Ltd.)

Higenamine is prohibited in sports as a β 2 -agonist by the World Anti-Doping Agency. As a key component of a great variety of plants, including the Annonaceae family, one aim of this research project was to evaluate whether the ingestion of Annona fruit could lead to higenamine adverse analytical findings. Single-dose administration studies including three Annona species (i.e., Annona muricata, Annona cherimola, and Annona squamosa) were conducted, leading to higenamine findings below the established minimum reporting level (MRL) of 10 ng/mL in urine. In consideration of c max values (7.8 ng/mL) observed for higenamine up to 24 h, a multidose administration study was also conducted, indicating cumulative effects, which can increase the risk of exceeding the applicable MRL doping after Annona fruit ingestion. In this study, however, the MRL was not exceeded at any time point. Further, the major urinary excretion of higenamine in its sulfo-conjugated form was corroborated, its stability in urine was assessed, and in the absence of reference material, higenamine sulfo-conjugates were synthesized and comprehensively characterized, suggesting the predominant presence of higenamine 7-sulfate. In addition, the option to include complementary biomarkers of diet-related higenamine intake into routine doping controls was investigated. A characteristic urinary pattern attributed to isococlaurine, reticuline, and a yet not fully characterized bismethylated higenamine glucuronide was observed after Annona ingestion but not after supplement use, providing a promising dataset of urinary biomarkers, which supports the discrimination between different sources of urinary higenamine detected in sports drug testing programs., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Psychedelic compounds have gained renewed interest for their potential therapeutic applications, but their metabolism and effects on complex biological systems remain poorly understood. Here, we present a systematic characterization of Lysergic Acid Diethylamide (LSD) metabolites in the model organism Caenorhabditis elegans using state-of-the-art analytical techniques. By employing ultra-high performance liquid chromatography coupled with high-resolution tandem mass spectrometry, we putatively identified a range of LSD metabolites, shedding light on their metabolic pathways and offering insights into their pharmacokinetics. Our study demonstrates the suitability of Caenorhabditis elegans as a valuable model system for investigating the metabolism of psychedelic compounds and provides a foundation for further research on the therapeutic potential of LSD., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Copaifera langsdorffii Desf. is recognised as one of most famous medicinal and economic species of Copaiba, occurring in several distinct biomes. An untargeted metabolomic approach was used to evaluate the chemical variability of C. langsdorffii from contrasting climates biomes (Atlantic Rainflorest and the semiarid Cerrado). Metabolomic analysis enabled the identification of 11 compounds, including glycosylated flavonoids and galloylquinic acid derivatives. Multivariate analysis highlighted that Cerrado population had a significantly higher concentrations of galloylquinic acid derivatives in comparison to the rainforest biome. Meanwhile, Atlantic Rainforest populations presented higher content of flavonols. Semiarid biome, reduced the concentration of flavonoids, mainly concerning quercetin and kaempferol derivatives, however, in this biome flavonoids were more diverse. Both chemical classes presented relevance to be used as geographical origin chemical markers by qualitative and quantitative features.

Blood transfusion is performed by cheating athletes to rapidly increase oxygen delivery to exercise muscles and enhance their performance. This method is banned by the World Anti-doping Agency (WADA). Heterologous or allogenic blood transfusion happens when blood from a different person is transfused. The method used to detect this type of doping is based on flow cytometry, by identifying variations in blood group minor antigens present on the red blood cells' surface. Transfusion practices have regained interest since the introduction of human recombinant erythropoietin detection method. It has been reported that the number of occurrences of two athletes sharing an identical phenotype in the same sport was five times higher than the theoretical populational probability. The present work describes the prevalence of 10 erythrocytes surface antigens in a population of 261 athletes from all five continents. The matching phenotype per sport is also described., (© 2022 John Wiley & Sons Ltd.)

Introduction: Maternal high-fat (HF) diet during gestation and lactation programs obesity in rat offspring associated with sex-dependent and tissue-specific changes of the endocannabinoid system (ECS). The ECS activation induces food intake and preference for fat as well as lipogenesis. We hypothesized that maternal HF diet would increase the lipid endocannabinoid levels in breast milk programming cannabinoid and dopamine signaling and food preference in rat offspring., Methods: Female Wistar rats were assigned into two experimental groups: control group (C), which received a standard diet (10% fat), or HF group, which received a high-fat diet (29% fat) for 8 weeks before mating and during gestation and lactation. Milk samples were collected to measure endocannabinoids and fatty acids by mass spectrometry. Cannabinoid and dopamine signaling were evaluated in the nucleus accumbens (NAc) of male and female weanling offspring. C and HF offspring received C diet after weaning and food preference was assessed in adolescence., Results: Maternal HF diet reduced the milk content of anandamide (AEA) ( p<0.05 ) and 2-arachidonoylglycerol (2-AG) ( p<0.05 ). In parallel, maternal HF diet increased adiposity in male ( p<0.05 ) and female offspring ( p<0.05 ) at weaning. Maternal HF diet increased cannabinoid and dopamine signaling in the NAc only in male offspring ( p<0.05 ), which was associated with higher preference for fat in adolescence ( p<0.05 )., Conclusion: Contrary to our hypothesis, maternal HF diet reduced AEA and 2-AG in breast milk. We speculate that decreased endocannabinoid exposure during lactation may induce sex-dependent adaptive changes of the cannabinoid-dopamine crosstalk signaling in the developing NAc, contributing to alterations in neurodevelopment and programming of preference for fat in adolescent male offspring., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dias-Rocha, Costa, Oliveira, Fassarella, Woyames, Atella, Santos, Pereira, Pazos-Moura, Almeida and Trevenzoli.)

In this study, a beetroot peel flour was made, and its in vitro antioxidant activity was determined in aqueous (BPFw) and ethanolic (BPFe) extracts. The influence of BPFw on breast cancer cell viability was also determined. A targeted betalain profile was obtained using high-resolution Q-Extractive Plus Orbitrap mass spectrometry (Obrtitrap-HRMS) alongside untargeted chemical profiling of BPFw using Ultra-High-Performance Liquid Chromatography with High-Resolution Mass Spectrometry (UHPLC-HRMS). BPFw and BPFe presented satisfactory antioxidant activities, with emphasis on the total phenolic compounds and ORAC results for BPFw (301.64 ± 0.20 mg GAE/100 g and 3032.78 ± 55.00 µmol T/100 g, respectively). The MCF-7 and MDA-MB-231 breast cancer cells presented reductions in viability when treated with BPFw, showing dose-dependent behavior, with MDA-MB-231 also showing time-dependent behavior. The chemical profiling of BPFw led to the identification of 9 betalains and 59 other compounds distributed amongst 28 chemical classes, with flavonoids and their derivates and coumarins being the most abundant. Three forms of betalain generated via thermal degradation were identified. However, regardless of thermal processing, the BPF still presented satisfactory antioxidant and anticancer activities, possibly due to synergism with other identified molecules with reported anticancer activities via different metabolic pathways.

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The metabolic fingerprint of a non-volatile fraction of Ocotea canaliculata (Rich.) Mez (Lauraceae) leaves was determined by UHPLC-HRMS analysis. Twenty-four compounds were suggestively identified by GNPS-FBMN. The results revealed a large production of flavonoids, mainly flavones and flavanones, a chemical class poorly described in the Ocotea genus. Within the identified compounds, four are being described for the first time in this genus. The major metabolite detected was astilbin, with a concentration corresponding to 23.2 ± 1.58% of the extracts. The expressive content of astilbin also highlights it as a chemical marker for the species. As a species that is classified as a complex, qualitative and semi-quantitative features obtained through the O. canaliculata flavonoid fingerprint can be further used for a more precise circumscription and species-specific characterization.

Coffee is one of the most consumed beverages worldwide. Cafestol is an endogenous coffee diterpene present in raw coffee beans and also found in hot beverages, with several biological activities. However, there is still little information on this molecule after ingestion of coffee infusion. Zebrafish (Danio rerio) is a promising in vivo model for metabolic studies due to the annotation of mammalian orthologs to encode enzymes related to drug metabolism. Experiments using Zebrafish Water Tank (ZWT) model produce more significant number of metabolites for molecular investigation in a cleaner matrix than other classical models, such as purified hepatocytes. This work aimed to investigate the biotransformation of cafestol by the ZWT model using ultra-performance liquid chromatography coupled to hybrid quadrupole-orbitrap high-resolution mass spectrometry equipped with electrospray ionization (UPLC-HRMS) supported by in silico approach using SMARTCyp, Way2Drug and XenoSite Softwares. Twenty-five metabolites of cafestol were proposed by in silico analysis, in which 5 phase I metabolites were confirmed in the ZWT by UPLC and MS/HRMS investigation: 6-hydroxy-cafestol, 6,12-dihydroxy-cafestol, 2-oxo-cafestol, 6-oxo-cafestol and one isomer whose position in the carboxyl group was not determined. These metabolites were observed during 9 h of the experiment, whose contents were associated with the behavioral responses of the fish., (Copyright © 2021. Published by Elsevier B.V.)

Freshwater sponges can be considered a promising new source of bioactive compounds for the pharmaceutical industry; however, the research on their chemical composition is still in the incipient stage. We evaluated the most endemic Amazonian freshwater sponge species from the Drulia and Metania genera by untargeted metabolomic approaches, based on UHPCL-HRMS, in order to identify chemical markers and explore the diversity of specialized metabolites. The use of untargeted approaches allowed us to observe subsets of metabolites that enabled the characterization of, not only each genus, but also, of each species. Freshwater sponge species presented themselves as rich sources of fatty acids and sterols, which were putatively identified. These metabolites were suggested as chemical markers for further targeted metabolomic studies.

Quinoline and 1,2,3-triazoles are well-known nitrogen-based heterocycles presenting diverse pharmacological properties, although their antileishmanial activity is still poorly exploited. As an effort to contribute with studies involving these interesting chemical groups, in the present study, a series of compounds derived from 4-aminoquinoline and 1,2,3-triazole were synthetized and biological studies using L. amazonensis species were performed. The results pointed that the derivative 4, a hybrid of 4-aminoquinoline/1,2,3-triazole exhibited the best antileishmanial action, with inhibitory concentration (IC 50 ) values of ~1 µM against intramacrophage amastigotes of L. amazonensis , and being 16-fold more active to parasites than to the host cell. The mechanism of action of derivative 4 suggest a multi-target action on Leishmania parasites, since the treatment of L. amazonensis promastigotes caused mitochondrial membrane depolarization, accumulation of ROS products, plasma membrane permeabilization, increase in neutral lipids, exposure of phosphatidylserine to the cell surface, changes in the cell cycle and DNA fragmentation. The results suggest that the antileishmanial effect of this compound is primarily altering critical biochemical processes for the correct functioning of organelles and macromolecules of parasites, with consequent cell death by processes related to apoptosis-like and necrosis. No up-regulation of reactive oxygen and nitrogen intermediates was promoted by derivative 4 on L. amazonensis -infected macrophages, suggesting a mechanism of action independent from the activation of the host cell. In conclusion, data suggest that derivative 4 presents selective antileishmanial effect, which is associated with multi-target action, and can be considered for future studies for the treatment against disease., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The detection and unambiguous identification of anabolic-androgenic steroid metabolites are essential in clinical, forensic, and antidoping analyses. Recently, sulfate phase II steroid metabolites have received increased attention in steroid metabolism and drug testing. In large part, this is because phase II steroid metabolites are excreted for an extended time, making them a potential long-term chemical marker of choice for tracking steroid misuse in sports. Comprehensive analytical methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), have been used to detect and identify glucuronide and sulfate steroids in human urine with high sensitivity and reliability. However, LC-MS/MS identification strategies can be hindered by the fact that phase II steroid metabolites generate nonselective ion fragments across the different metabolite markers, limiting the confidence in metabolite identifications that rely on exact mass measurement and MS/MS information. Additionally, liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is sometimes insufficient at fully resolving the analyte peaks from the sample matrix (commonly urine) chemical noise, further complicating accurate identification efforts. Therefore, we developed a liquid chromatography-ion mobility-high resolution mass spectrometry (LC-IM-HRMS) method to increase the peak capacity and utilize the IM-derived collision cross section (CCS) values as an additional molecular descriptor for increased selectivity and to improve identifications of intact steroid analyses at low concentrations.

Knowledge of the metabolic profile is essential for doping control analysis in sport since most drugs are excreted after an elaborate biotransformation process. Currently, Zebrafish Water Tank (ZWT) model has been applied to investigate the metabolism of different doping agents. Nevertheless, the class of glucocorticoids has not been subjected to this model for metabolism studies. In the present work, budesonide (BUD) was applied as a pilot to investigate the metabolic pathways of glucocorticoids in the ZWT model. The BUD biotransformation in ZWT model was compared to the described metabolism in humans. Samples from ZWT experiments were collected after BUD administration and analyzed by Liquid Chromatography coupled to High Resolution Mass Spectrometry (LC-HRMS). Following the identification and characterization of all significant metabolites described for humans, it was observed that the ZWT was able to produce in a relevant amount the main target for doping control purposes: the 6β-hydroxy BUD. In addition, prior knowledge about the lack of butyrylcholinesterase activity in the zebrafish organism was considered for the evaluation for the formation of the 16α-hydroxy prednisolone, the most intense BUD metabolite in human urine. Biotransformation of BUD by ZWT focused on metabolites with the acetal fraction preserved, including the intermediate metabolite for the 16α-hydroxy prednisolone pathway. However,analternative metabolic pathway for the complete biotransformation of the 16α-hydroxy prednisolone intermediate was not observed, leading to the absence of the major human metabolite in the ZWT model. The findings reported in this study elucidate for the first time the application and limitations of the ZWT model to evaluate the metabolism of other glucocorticoids., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Untargeted metabolomics is a powerful tool in chemical fingerprinting. It can be applied in phytochemistry to aid species identification, systematic studies and quality control of bioproducts. This approach aims to produce as much chemical information as possible, without focusing on any specific chemical class, thus, requiring extensive chemometric effort. This study aimed to evaluate the feasibly of an untargeted metabolomics method in phytochemistry by a study case of the Copaifera genus (Fabaceae). This genus contains significant medicinal species used worldwidely. Copaifera exploitation issues include a lack of chemical data, ambiguous species identification methods and absence of quality control for its bioproducts. Different organs of five Copaifera species were analysed by UHPLC-HRMS/MS, GNPS platform and chemometric tools. Untargeted metabolomics enabled the identification of 19 chemical markers and 29 metabolites, distinguishing each sample by species, plant organs, and biome type. Chemical markers were classified as flavonoids, terpenoids and condensed tannins. The applied method provided reliable information about species chemodiversity using fast workflow with little sampling size. The untargeted approach by UHPLC-HRMS/MS proved to be a promising tool for species identification, pharmacological prospecting and in the future for the quality control of extracts used in the manufacture of bioproducts., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Stanozolol (STAN) is an androgen anabolic steroid often misused in sports competitions and prohibited at all times by the World Anti-Doping Agency (WADA). It can be long term detected by the analysis of human urine for traces of intact glucuronide metabolites. The Zebrafish Water Tank (ZWT) experimental setup can produce phase I STAN metabolites. In the present study, we investigated the in vivo phase II metabolism of STAN through the ZWT model to determine whether the ZWT produces metabolites relevant for doping control. We added STAN to a 200 mL recipient containing eight fish at 32 ± 1 °C. We analyzed the noninvasive samples (recipient water) both with and without pretreatment using Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC-HRMS/MS) in positive ionization mode. Our data show that four hydroxylated-sulfate and four hydroxylated-glycoconjugate metabolites were formed, two of the last ones being 3'OH-STAN-Glucuronide and 16β-OH-STAN-Glucuronide. Additionally, two STAN-Glucuronide derivatives were produced: one was confirmed to be 17epi-STAN-N-Glucuronide, and the other was presumed to be STAN-O-Glucuronide. After eight hours of the experiment, STAN-O-Glucuronide was the most intense phase II metabolite produced. The accumulation curves suggest that high concentrations of fish and substrate in water are required to form phase II metabolites., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Dermorphin is a heptapeptide with an analgesic potential higher than morphine that does not present the same risk for the development of tolerance. These pharmacological features make dermorphin a potential doping agent in competitive sports and it is already prohibited for racehorses. For athletes, the development of an efficient strategy to monitor for its abuse necessitates an investigation of the metabolism of dermorphin in humans., Methods: Here, human liver microsomes and zebrafish were utilized as model systems of human metabolism to evaluate the presence and kinetics of metabolites derived from dermorphin. Five hours after its administration, the presence of dermorphin metabolites could be detected in both models by liquid chromatography coupled to highresolution mass spectrometry., Results: Although the two models showed common results, marked differences were also observed in relation to the formed metabolites. Six putative metabolites, based on their exact masses of m/z 479.1915, m/z 501.1733, m/z 495.1657, m/z 223.1073, m/z 180.1017 and m/z 457.2085, are proposed to represent the metabolic pattern of dermorphin. The major metabolite generated from the administration of dermorphin in both models was YAFG-OH (m/z 457.2085), which is the N-terminal tetrapeptide previously identified from studies on rats., Conclusion: Its extensive characterization and commercial availability suggest that it could serve as a primary target analyte for the detection of dermorphin misuse. The metabolomics approach also allowed the assignment of other confirmatory metabolites., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Dermorphin is a peptide with analgesic actions similar to morphine, but with greater effect and less potential to cause tolerance. The use of dermorphin has been documented in race horses, and its use in humans has already been reported. Considering the potential advantages from the use of dermorphin over morphine, a method to monitor it, and its main metabolite dermorphin (1-4) in humans becomes necessary for doping control. Here, we present two orthogonal methods for this purpose: a high-throughput liquid chromatography coupled to high-resolution mass spectrometry (HRMS) as an initial testing procedure and liquid chromatography-tandem mass spectrometry (MS/MS) in the selected reaction monitoring (SRM) acquisition mode for a confirmation procedure. For urine samples, pretreatment through a mixed-mode weak cation-exchange solid-phase extraction emerged as an effective approach to extract peptides from the biological sample. For the HRMS analysis, a full-MS scan acquisition mode was selected to detect the exact masses of dermorphin and dermorphin (1-4) at m/z 803.37226 and 457.20816, respectively. The SRM method used in the MS/MS confirmation protocol presented high specificity and sensitivity. The selected product ions for dermorphin were 602.2, 202.1 and 574.3 and for dermorphin (1-4) were 207.1, 223.1, and 235.1. Both methods were evaluated for specificity, repeatability, carryover, matrix effects, and recovery. No carryover and matrix effects were detected. The limit of detection for initial testing procedure and the limit of identification for confirmation procedure was 2.5 ng/ml. Also, specificity and robustness were acceptable for the application. Together, the developed methods proved to be efficient for the analysis of dermorphin and metabolite for human doping control purpose., (© 2020 John Wiley & Sons, Ltd.)

Testolactone is a potent steroid aromatase (CYP19A1) inhibitor, and its main effect is a reduction in estradiol and estrone and an increase in testosterone and androstenedione levels. In this work, we evaluated a zebrafish water tank (ZWT) as a model to investigate testolactone biotransformation and the possibility to increase knowledge regarding the applicability of the ZWT on steroid hormone elimination research, as well as on the impact of steroid hormones on the endogenous metabolism of zebrafish. High-resolution mass spectrometry combined with SIEVE software was used to discriminate the peaks of interest based on significant changes in the relative signal intensity of the m/z values between different ZWT experiments. The metabolites, 4,5-dihydrotestolactone and 1,2,4,5-tetrahydrotestolactone, the same metabolites as those described in humans, were detected in ZWT, both in quite similar proportions. The presence of testolactone in the ZWT caused a rise in testosterone and androstenedione in the water tank, similar to that in human serum. These data suggest that, while the concentration of testolactone was high enough to inhibit the aromatase enzyme, an accumulation of androgens in the water occurred, indicating that the ZWT can be considered a model to investigate the impact of steroids on live organisms.

Untargeted metabolomics aim to provide a global chemical fingerprint of biological matrices. This research field can be used in phytochemical screenings for bioactive species or in the identification of species. Despite its importance in providing a global chemical profile, little research has focused on the optimization of the extraction methods, as each type of matrix requires a specific procedure. Therefore, we propose to evaluate the effect of different extraction features in an ultrasound-assisted extraction for the untargeted metabolomic study of an Ocotea species, a genus of great economical interest but little chemical exploitation. Method optimization was performed in a full factorial 2 2 3 2 design, evaluating the solvent composition, extraction temperature, sample particle size and sample : solvent ratio effects in the metabolomic response. The effect of these parameters on the quality of the untargeted metabolomic profiles was studied by analysis of the extraction yield as well as the chromatographic and spectrometric profiles. Most substances identified were glycosylated flavonoids and aporphinic alkaloids. The application of 70% ethanol enhanced the extraction of several specialized metabolites. Statistical analysis of extraction yield and chemical profiles indicates that high temperatures and low proportion between sample and extracting solvent reduce the quality and modify the chemical profile, both qualitatively and quantitatively. The use of 70% ethanol as the extracting solvent, 1 : 12 sample : solvent ratio, 40 °C as the extraction temperature and particle size of 0.595 mm were the optimized conditions to produce a comprehensive chemical profile for Ocotea guianensis ., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background: Approximately, 5-7 million people are infected with T. cruzi in the world, and approximately 10,000 people per year die of complications linked to this disease., Methods: This work describes the construction of a new family of hidrazonoyl substituted derivatives, structurally designed exploring the molecular hybridization between megazol and nitrofurazone., Results and Discussion: The compounds were evaluated for their in vitro activity against bloodstream trypomastigotes of Trypanosoma cruzi, etiological agent of Chagas disease, and for their potential toxicity to mammalian cells., Conclusion: Among these hydrazonoyl derivatives, we identified the derivative (4) that showed trypanocidal activity (IC50/24 h = 15.0 µM) similar to Bz, the standard drug, and low toxicity to mammalian cells, reaching an SI value of 18.7., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Carbon isotope ratio (CIR) confirmation is one of the most complex and delicate analyses in the doping control field, due to the nature of the molecules to be confirmed, normally present in urinary samples as a consequence of an endogenous production. The requirements for method validation established by the World Anti-Doping Agency (WADA) have been pushing the accredited laboratories to improve their methods. The choice of the method is always a cost benefit ratio involving a hard-working and time-consuming analysis and the guarantee of reporting of reliable results. This work presents the method fully validated by the Brazilian Doping Control Laboratory as part of the preparation for the Rio de Janeiro Summer Olympic and Paralympic Games 2016. Sample preparation encompassed solid-phase extraction, liquid-liquid extraction, enzymatic hydrolysis, acetylation, and purification by preparative high-performance liquid chromatography, and analyses were performed by gas chromatography/combustion/isotope ratio mass spectrometry. This proved to be a robust method to CIR confirmation in a big event, as demonstrated by the analysis of 179 samples during the Games 2016, from clearly negative results and adverse findings for testosterone (T) and related substances, boldenone and its metabolite, 19-norandrosterone and formestane. Two atypical findings were also reported for T and metabolites.

Sibutramine is cited by the World Anti-Doping Agency as a stimulant. According to the literature, sibutramine is extensively metabolized into N-desmethyl-sibutramine (M1), N-bisdesmethyl-sibutramine (M2) and monohydroxy derivatives of M1 and M2. Therefore, it is important to verify new sibutramine metabolites through current analytical methodologies, such as liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Furthermore, the development of a comprehensive approach to investigate sibutramine metabolism can increase the detection window for stimulant misuse and enable advancements in pharmacological studies. This work aimed to develop and evaluate the performance of an LC-HRMS method applying Design of Experiments (DoE) for sibutramine metabolite analysis in human urine. After optimizing the method by DoE, the final chromatographic conditions were based on reversed-phase chromatography using a C18 column with a ramp time of 25 min, a flow rate of 0.17 mL min -1 and a temperature of 50 °C. Mobile phase A consisted of water with 0.1% formic acid and 5 mM ammonium formate, and mobile phase B consisted of methanol with 0.1% formic acid; the initial gradient percent was 15% B, and the injection volume was 5 μL. In addition to the hydroxylated metabolites previously described in human urine, dihydroxy derivatives of M1 and M2 were observed for the first time. These dihydroxy derivative metabolites can be applied as new targets for doping control., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Zebrafish has become a popular model organism in several lines of biological research sharing physiological, morphological and histological similarities with mammals. In fact, many human cytochrome P450 (CYP) enzymes have direct orthologs in zebrafish, suggesting that zebrafish xenobiotic metabolic profiles may be similar to those in mammals. The focus of the review is to analyse the studies that have evaluated the metabolite production in zebrafish over the years, either of the drugs themselves or xenobiotics in general (environmental pollutants, natural products, etc.), bringing a vision of how these works were performed and comparing, where possible, with human metabolism. Early studies that observed metabolic production by zebrafish focused on environmental toxicology, and in recent years the main focus has been on toxicity screening of pharmaceuticals and drug candidates. Nevertheless, there is still a lack of standardization of the model and the knowledge of the extent of similarity with human metabolism. Zebrafish screenings are performed at different life stages, typically being carried out in adult fish through in vivo assays, followed by early larval stages and embryos. Studies comparing metabolism at the different zebrafish life stages are also common. As with any non-human model, the zebrafish presents similarities and differences in relation to the profile of generated metabolites compared to that observed in humans. Although more studies are still needed to assess the degree to which zebrafish metabolism can be compared to human metabolism, the facts presented indicate that the zebrafish is an excellent potential model for assessing xenobiotic metabolism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The number of substances nominally listed in the prohibited list of the World Anti-Doping Agency increases each year. Moreover, many of these substances do not have a single analytical target and must be monitored through different metabolites, artifacts, degradation products, or biomarkers. A new analytical method was developed and validated for the simultaneous analysis of peptides and organic molecules using a single sample preparation and LC-Q-HRMS detection. The simultaneous analysis of 450 target molecules was performed after cleanup on a mixed-mode solid-phase extraction cartridge, combined with untreated urine. The cleanup solvent and reconstitution solvent were the most important parameters for achieving a comprehensive sample preparation approach. A fast chromatographic run based on a multistep gradient was optimized under different flows; the detection of all substances without isomeric coelution was achieved in 11 minutes, and the chromatographic resolution was considered a critical parameter, even in high-resolution mass spectrometry detection. The mass spectrometer was set to operate by switching between positive and negative ionization mode for FULL-MS, all-ion fragmentation, and FULL-MS/MS 2 . The suitable parameters for the curved linear trap (c-trap) conditions were determined and found to be the most important factors for the development of the method. Only FULL-MS/MS 2 enables the detection of steroids and peptides at concentrations lower than the minimum required performance levels set by World Anti-Doping Agency (1 ng mL -1 ). The combination of the maximum injection time of the ions into the c-trap, multiplexing experiments, and loop count under optimized conditions enabled the method to be applied to over 10 000 samples in only 2 months during the 2016 Rio Summer Olympic and Paralympic Games. The procedure details all aspects, from sample preparation to mass spectrometry detection. FULL-MS data acquisition is performed in positive and negative ion mode simultaneously and can be applied to untargeted approaches., (Copyright © 2018 John Wiley & Sons, Ltd.)

Cancer is one of the biggest problems in public health worldwide. Plants have been shown important role in anticancer research. Viscum album L. (Santalaceae), commonly known as mistletoe, is a semi-parasitic plant that grows on different host trees. In complementary medicine, extracts from European mistletoe ( Viscum album L.) have been used in the treatment of cancer. The study was conducted to identify chemical composition and antitumor potential of Viscum album tinctures. Chemical analysis performed by high resolution chromatography equipped with high resolution mass spectrometer identified caffeic acid, chlorogenic acid, sakuranetin, isosakuranetin, syringenin 4-O-glucoside, syringenin 4-O-apiosyl-glucoside, alangilignoside C and ligalbumoside A compounds. Some of these compounds are probably responsible for the reduction of tumoral cellular growth in a dose-dependent manner. It was observed that melanoma murine cells (B16F10) were more sensitive to V. album tinctures than human leukaemic cells (K562), besides non-tumoral cells (MA-104) had a much lower cytotoxicity to them. Apoptotic-like cells were observed under light microscopy and were confirmed by a typical DNA fragmentation pattern. Additionally, flow cytometry results using Annexin-V/FITC permitted to quantify increased expression of early and late apoptotic markers on tumoral cells, confirming augmented Sub G0 population, which was probably associated with a consistent decrease in G1, and an increase in S or G2/M populations. Results indicate the chemical composition of V. album tinctures influences the mechanisms of in vitro tumoral cell death, suggesting a potential use in cancer pharmacotherapy research.

This paper summarises the results obtained from the doping control analyses performed during the Summer XXXI Olympic Games (August 3-21, 2016) and the XV Paralympic Games (September 7-18, 2016). The analyses of all doping control samples were performed at the Brazilian Doping Control Laboratory (LBCD), a World Anti-Doping Agency (WADA)-accredited laboratory located in Rio de Janeiro, Brazil. A new facility at Rio de Janeiro Federal University (UFRJ) was built and fully operated by over 700 professionals, including Brazilian and international scientists, administrative staff, and volunteers. For the Olympic Games, 4913 samples were analysed. In 29 specimens, the presence of a prohibited substance was confirmed, resulting in adverse analytical findings (AAFs). For the Paralympic Games, 1687 samples were analysed, 12 of which were reported as AAFs. For both events, 82.8% of the samples were urine, and 17.2% were blood samples. In total, more than 31 000 analytical procedures were conducted. New WADA technical documents were fully implemented; consequently, state-of-the-art analytical toxicology instrumentation and strategies were applied during the Games, including different types of mass spectrometry (MS) analysers, peptide, and protein detection strategies, endogenous steroid profile measurements, and blood analysis. This enormous investment yielded one of the largest Olympic legacies in Brazil and South America. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

One of the greatest challenges in anti-doping science is the large number of substances available and the difficulty in finding the best analytical targets to detect their misuse. Therefore, metabolism studies involving prohibited substances are fundamental. However, metabolism studies in humans could face an important ethical bottleneck, especially for non-approved substances. An emerging model for metabolism assessment is the zebrafish, due to its genetic similarities with humans. In the present study, the ability of adult zebrafish to produce metabolites of sibutramine and stanozolol, substances with a well-known metabolism that are widely used as doping agents in sports, was evaluated. They represent 2 of the most abused classes of doping agents, namely, stimulants and anabolic steroids. These are classes that have been receiving attention because of the upsurge of synthetic analogues, for which the side effects in humans have not been assessed. The samples collected from the zebrafish tank water were hydrolysed, extracted by solid-phase extraction, and analysed by liquid chromatography with high resolution mass spectrometry (LC-HRMS). Adult zebrafish could produce several sibutramine and stanozolol metabolites, including demethylated, hydroxylated, dehydroxylated, and reduced derivatives, all of which have already been detected in human urine. This study demonstrates that adult zebrafish can absorb, oxidise, and excrete several metabolites in a manner similar to humans. Therefore, adult zebrafish seem to be a very promising tool to study human-like metabolism when aiming to find analytical targets for doping control. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)