Comparative study of sample preparation procedures to determine the main compounds in ayahuasca beverages by QuEChERS and high-performance liquid chromatography analysis.

INTRODUCTION: Ayahuasca is a psychoactive drink originally consumed by indigenous people of the Amazon. The lack of regulation of this drink leads to uncontrolled consumption, and it is often consumed in religious contexts. OBJECTIVE: The aim of this work is to compare three miniaturised extraction techniques for extracting the main ayahuasca compounds from beverages. METHODOLOGY: Three sample pretreatment techniques were evaluated (dispersive liquid-liquid microextraction [DLLME], microextraction by packed sorbent [MEPS] and QuEChERS [Quick, Easy, Cheap, Effective, Rugged and Safe]) for the simultaneous extraction of N,N-dimethyltryptamine (DMT), tetrahydroharmine (THH), harmine, harmaline, harmol and harmalol from ayahuasca beverage samples. Then, the most promising technique (QuEChERS) was chosen to pre-concentrate the analytes, subsequently detected by high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD). RESULTS: The procedure was optimised, with the final conditions being 500 µL of extractor solvent, 85 mg of primary secondary amine (PSA) and 4 s of vortexing. The analytical method was validated, showing to be linear between 0.16 and 10 µg/mL for ß-carbolines and between 0.016 and 1 µg/mL for DMT, with coefficients of determination (R2) between 0.9968 and 0.9993. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.16 µg/mL for all compounds, except for DMT (0.016 µg/mL) and extraction efficiencies varied between 60.2% and 88.0%. CONCLUSION: The analytical methodology proved to be accurate and precise, with good linearity, LODs and LLOQs. This method has been fully validated and successfully applied to ayahuasca beverage samples.

Characterisation of Cannabis-Based Products Marketed for Medical and Non-Medical Use Purchased in Portugal.

Cannabis-based products have gained attention in recent years for their perceived therapeutic benefits (with cannabinoids such as THC and CBD) and widespread availability. However, these products often lack accurate labelling regarding their cannabinoid content. Our study, conducted with products available in Portugal, revealed significant discrepancies between label claims and actual cannabinoid compositions. A fully validated method was developed for the characterisation of different products acquired from pharmacies and street shops (beverages, herbal samples, oils, and cosmetic products) using high-performance liquid chromatography coupled with a diode array detector. Linearity ranged from 0.4 to 100 µg/mL (0.04-10 µg/mg) (THC, 8-THC, CBD, CBG, CBDA, CBGA), 0.1-100 µg/mL (0.01-10 µg/mg) (CBN), 0.4-250 µg/mL (0.04-25 µg/mg) (THCA-A), and 0.8-100 µg/mL (0.08-10 µg/mg) (CBCA). Among sampled beverages, none contained detectable cannabinoids, despite suggestive packaging. Similarly, oils often differed from the declared cannabinoid compositions, with some containing significantly higher CBD concentrations than labelled. These inconsistencies raise serious concerns regarding consumer safety and informed decision-making. Moreover, our findings underscore the need for stringent regulation and standardised testing protocols to ensure the accuracy and safety of cannabis-based products.

Effect of fluralaner on the biology, survival, and reproductive fitness of the neotropical malaria vector Anopheles aquasalis.

BACKGROUND: Reducing mosquito abundance or interfering with its ability to support the parasite cycle can help to interrupt malaria in areas of significant risk of malaria transmission. Fluralaner is a safe and effective drug for veterinary use indicated for the treatment against fleas and ticks which acts as an antagonist of chloride ion channels mediated by γ-aminobutyric acid (GABA), preventing the entry of these ions into the postsynaptic neuron, leading to hyperexcitability of the postsynaptic neuron of the central nervous system of arthropods. Fluralaner demonstrated insecticidal activity against different insect species. METHODS: The study aimed to evaluate the effects of fluralaner on the biology, survival, and reproductive fitness of Anopheles aquasalis. The following lethal concentrations (LC) were determined for An. aquasalis: LC5 = 0.511 µM; LC25 = 1.625 µM; LC50 = 3.237 µM. RESULTS: A significant decrease (P < 0.001) was evident in the number of eggs, larvae, and pupae in the group exposed to a sublethal dose of fluralaner when compared to a control group (without the drug). Using blood from dogs after administration of fluralaner, it was observed that the drug causes 100% mortality in An. aquasalis in less than 24 h after feeding; this effect remains even after 90 days in all samples. DISCUSSION: Fluralaner showed the same result for up to 60 days, and after that, there was a slight reduction in its effect, evidenced by a decrease in the percentage of dead females; however, still significant when compared to the control group. CONCLUSION: Fluralaner affects the biology and reduction of survival in An. aquasalis in a lasting and prolonged period, and its fecundity with lower dosages, is a strong candidate for controlling disease vectors.

Chemical composition, antioxidant, and antimicrobial activities of six commercial essential oils.

Essential oils (EOs) and their components extracted from medicinal and aromatic plants are used in several areas, such as perfumery and chemical, cosmetic, food, and pharmaceutical industries. Considering the different applications of EOs, this work aimed to screen the composition and the bioactivities properties of the EOs of Foeniculum vulgare, Helichrysum stoechas, Mentha pulegium, Pinus pinaster, Ruta graveolens, and Thymus mastichina. Gas chromatography-mass spectrometry revealed the presence of different compounds in EOs F. vulgare (12), H. stoechas (27), M. pulegium (8), P. pinaster (24), R. graveolens (8), and T. mastichina (16). All the EOs showed antioxidant activity acting through inhibition of lipid peroxidation, while only two EOs (H. stoechas and M. pulegium) scavenged the free radicals of DPPH. Mentha pulegium and T. mastichina EOs showed the strongest antimicrobial activity. Also, the effect on the fibroblast's viability was directly proportional to the EOs concentration, and the highest cytotoxic effect was registered with R. graveolens EO. The present study revealed significant bioactive properties of different EOs, highlighting M. pulegium and T. mastichina EOs to be considered in further studies for potential use in the food and pharmaceutical industries, due to their antioxidant and antimicrobial properties.

Perceptions, feelings, and the routine of older adults during the isolation period caused by the COVID-19 pandemic: a qualitative study in four countries.

OBJECTIVE: To evaluate the knowledge, routine, and perception of older adults from four countries about dealing with COVID-19 in the social isolation period. METHODS: Qualitative study with semistructured interviews. Older persons (≥60 years old), who lived in the urban centers of four different countries (Brazil, United States, Italy, and Portugal), were asked about the changes experienced during the pandemic, new habits or restrictions during isolation, sources of social and emotional support, and their knowledge about COVID-19 pandemic. Data was transcribed, codified, and submitted to content analysis. RESULTS: Twenty-five older persons (10 from Brazil, 5 from Italy, 5 from Portugal, and 5 from the United States) were interviewed. Participants reported feeling restricted in their daily life activities and emotional instability. Described adaptations in habits, coping strategies, and greater understanding of the diseased based on information available in the media. There was homogeneity in the statements of the elderly, showing that the pandemic affected them in a similar way, even though they lived in different cultures and contexts. CONCLUSION: The social isolation caused by the COVID-19 pandemic changed the structure for the performance of many occupations, having an impact in the perception social participation and wellbeing of elders. This data can aid health professionals to outline strategies to deal with the impact of the social isolation in older persons.

Natural Products: Therapeutic Properties and Beyond II.

Historically, natural products have contributed to drug discovery as a source of active molecules due to their great diversity and structural complexity [...].

Evaluation of the In Vitro Wound-Healing Potential of Ayahuasca.

Ayahuasca is an Amazonian drink, which contains ß-carboline alkaloids and N,N-dimethyltryptamine. The aim of this study was to evaluate the healing potential of decoctions of a commercial mixture, four individual plants and four mixtures of two plants used in the ayahuasca preparation. Thus, the cytotoxic potential of the samples was evaluated and a wound-healing assay was performed with a NHDF cell line. Subsequently, a parallel artificial membrane permeability assay was also performed, to verify if any psychoactive compound could be absorbed by skin fibroblasts. The integrity and permeability of the cell layer were also evaluated, using the transepithelial electrical resistance assay and Lucifer yellow permeability assay, respectively. The compounds absorbed by the cell layer were quantified by high-performance liquid chromatography coupled to a diode array detector. The results showed that only one sample showed cytotoxicity and all the others promoted the migration of skin fibroblasts. Additionally, it was also verified that ß-carbolynic alkaloids and N,N-dimethyltriptamine were not absorbed by the cell layer, and in general, did not interfere with its permeability and integrity. To the best of our knowledge, this is the first study where ayahuasca's wound-healing potential was evaluated.

Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 Detection.

The fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy. The novelty of this work lies in the design and optimization of two MBs for detection of SARS-CoV-2, namely, concentration, fluorescence plateaus of hybridization, reaction temperature and real-time results. We also identify putative G-quadruplex (G4) regions in the genome of SARS-CoV-2. A total of 458 nasopharyngeal and throat swab samples (426 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity, and specificity. Our results show that the samples with higher fluorescence levels correspond to those with low Ct values, suggesting a correlation between viral load and increased MB fluorescence. The proposed assay represents a fast (total duration of 2 h 20 min including amplification and fluorescence reading stages) and simple way of detecting SARS-CoV-2 in clinical samples from the upper respiratory tract.

Psychoactive Substances of Natural Origin: Toxicological Aspects, Therapeutic Properties and Analysis in Biological Samples.

The consumption of new psychoactive substances (NPSs) has been increasing, and this problem affects several countries worldwide. There is a class of NPSs of natural origin, consisting of plants and fungi, which have a wide range of alkaloids, responsible for causing relaxing, stimulating or hallucinogenic effects. The consumption of some of these substances is prompted by religious beliefs and cultural reasons, making the legislation very variable or even ambiguous. However, the abusive consumption of these substances can present an enormous risk to the health of the individuals, since their metabolism and effects are not yet fully known. Additionally, NPSs are widely spread over the internet, and their appearance is very fast, which requires the development of sophisticated analytical methodologies, capable of detecting these compounds. Thus, the objective of this work is to review the toxicological aspects, traditional use/therapeutic potential and the analytical methods developed in biological matrices in twelve plant specimens (Areca catechu, Argyreia nervosa, Ayahuasca, Catha edulis, Datura stramonium, Lophophora williamsii, Mandragora officinarum, Mitragyna speciosa, Piper methysticum Forst, Psilocybe, Salvia divinorum and Tabernanthe iboga).

In Vitro Study of the Bioavailability and Bioaccessibility of the Main Compounds Present in Ayahuasca Beverages.

Ayahuasca is a psychoactive beverage that contains the psychoactive compound N,N-dimethyltryptamine and ß-carboline alkaloids. This study aims at determining in vitro the bioavailability and bioaccessibility of the main compounds present in decoctions of four individual plants, in a commercial mixture and in four mixtures of two individual plants used in the preparation of Ayahuasca. The samples were subjected to an in vitro digestion process, and the Caco-2 cell line was used as an absorption model. The integrity and permeability of the cell monolayer were evaluated, as well as the cytotoxicity of the extracts. After digestion and cell incubation, the compounds absorbed by the cell monolayer were quantified by high-performance liquid chromatography coupled to a diode array detector. The results showed that compounds such as N,N-dimethyltryptamine, Harmine, Harmaline, Harmol, Harmalol and Tetrahydroharmine were released from the matrix during the in vitro digestion process, becoming bioaccessible. Similarly, some of these compounds, after being incubated with the cell monolayer, were absorbed, becoming bioavailable. The extracts did not show cytotoxicity after cell incubation, and the integrity and permeability of the cell monolayer were not compromised.

Correction: Simão, A.Y., et al. Evaluation of the Cytotoxicity of Ayahuasca Beverages. Molecules 2020, 25, 5594.

This paper [...].

Sweet Cherry Extract Targets the Hallmarks of Cancer in Prostate Cells: Diminished Viability, Increased Apoptosis and Suppressed Glycolytic Metabolism.

The present work evaluated the anticancer properties of sweet cherry (Prunus avium) extract on human prostate cells. Several sweet cherry cultivars from Fundão (Portugal) were methanol-extracted and their phytochemical composition characterized. The Saco "late harvest" extract was highly-enriched in anthocyanins and selected for use in biological assays. Non-neoplastic (PNT1A) and neoplastic (LNCaP and PC3) human prostate cells were treated with 0-2,000 µg/ml of extract for 48-96 h. Cell viability was evaluated by the MTT assay. Apoptosis, oxidative stress, and glycolytic metabolism were assessed by Western blotting and enzymatic assays. Glucose consumption and lactate production were measured spectrophotometrically. Saco cherry extract diminished the viability of neoplastic and non-neoplastic cells, whereas enhancing apoptosis in LNCaP. Cherry extract-treatment also diminished oxidative damage and suppressed glycolytic metabolism in LNCaP cells. These findings widened the knowledge on the mechanisms by which cherry extract modulate cell physiology, demonstrating their broad action over the hallmarks of cancer.

Phytochemical Characterization, Bioactivities Evaluation and Synergistic Effect of Arbutus unedo and Crataegus monogyna Extracts with Amphotericin B.

The increased resistance to drugs by pathogens is a serious problem, with plants showing to be promising sources for the development of new drugs or the improvement of the effect of existing antimicrobial agents. Considering this, we aimed to evaluate the bioactivities of Arbutus unedo and Crataegus monogyna. Thus, the leaves were first extracted with methanol and then fractionated with different solvents. Phenolic compound profiles were assessed by HPLC-PDA-MSn and the antioxidant activity was evaluated using DPPH method and ß-carotene bleaching assay. The antimicrobial activity of extracts was tested against several microorganisms. A. unedo contained mainly galloyl esters, hydrolysable tannins, and flavonoids, while in C. monogyna, 5-caffeoylquinic acid and flavonoids were the most representative polyphenols. Crude extracts showed antioxidant activity and the extracts and fractions displayed a weak antibacterial activity; however inhibiting the growth of Candida tropicalis and C. lusitaniae to which A. unedo extract showed higher activity. Most of the extracts and fractions demonstrated synergistic or additive interactions with amphotericin B against Candida spp. Therefore, the present study revealed significant bioactive properties of the extracts and fractions of A. unedo and C. monogyna, such as antioxidant and antifungal activities.

Julbernardia paniculata and Pterocarpus angolensis: From Ethnobotanical Surveys to Phytochemical Characterization and Bioactivities Evaluation.

Julbernardia paniculata and Pterocarpus angolensis are two plant species with important application in African traditional medicine, particularly in Angola, in the treatment of several diseases. However, scientific studies concerning these species are scarce. The goal of this work was to know better which medicinal approaches are used by the Huíla population in Angola by means of ethnobotanical surveys. Furthermore, extracts of both plants were phytochemically characterized. Antioxidant, anti-inflammatory, wound-healing activities, and potential cytotoxicity were also studied. With this study it was possible to verify that 67% of the individuals that use medicinal plants are women, and their main therapeutic uses are the treatment of problems of the digestive system and skin disorders. Barks of J. paniculata and leaves of P. angolensis are the most often used plant parts. Through high-performance liquid chromatography coupled to diode-array detector (HPLC-DAD) and GC-MS it was possible to characterize the chemical composition of the two species, which are rich in phenolic compounds, terpenes, terpenoids, sesquiterpenoids and fatty acids. Both plants showed to possess antioxidant, anti-inflammatory proprieties, and wound-healing activity. To the best of our knowledge, this is the most comprehensive study of these two species and the first ethnobotanical and ethnopharmacological study of medicinal plants from this region of Angola.

Evaluation of the Cytotoxicity of Ayahuasca Beverages.

Ayahuasca is a beverage consumed at shamanic ceremonies and currently has gained popularity on recreational scenarios. It contains beta-carboline alkaloids and N,N-dimethyltryptamine, which possesses hallucinogenic effects. Only a few studies have elicited the psychoactive effects and the dose of such compounds on neurological dopaminergic cells or animals. In this work, we aimed to study the cytotoxic effects of these compounds present in ayahuasca beverages and on five different teas (Banisteriopsis caapi, Psychotria viridis, Peganum harmala, Mimosa tenuiflora and Dc Ab (commercial name)) preparations on dopaminergic immortalized cell lines. Moreover, a characterization of the derivative alkaloids was also performed. All the extracts were characterized by chromatographic systems and the effect of those compounds in cell viability and total protein levels were analyzed in N27 dopaminergic neurons cell line. This is the first article where cytotoxicity of ayahuasca tea is studied on neurological dopaminergic cells. Overall, results showed that both cell viability and protein contents decreased when cells were exposed to the individual compounds, as well as to the teas and to the two mixtures based on the traditional ayahuasca beverages.

Socio-economic and demographic characteristics associated with risk behaviour patterns for chronic non-communicable diseases in Brazil: data from the National Health Survey, 2013.

OBJECTIVE: To identify risk behaviour patterns for chronic non-communicable diseases (NCD) in the Brazilian population and to investigate associated socio-economic and demographic factors. DESIGN: Factor analysis was used to identify patterns considering the following risk behaviours: consumption of soft drinks/artificial juice, sweet foods, red meat with apparent fat, chicken skin; inadequate consumption of fruits and vegetables; alcohol abuse; smoking; absence of physical activity during leisure time; and time spent watching television. The χ 2 test was used to compare ratios. All analyses accounted for weighting factors and the study's complex sampling design effect. The socio-economic and demographic variables evaluated were gender, age, schooling level and macro region of residence. SETTING: National Health Survey, a household survey with national representation, conducted in 2013 in Brazil.ParticipantsIndividuals (n 60202) aged 18 years or over. RESULTS: Four risk behaviour patterns were identified: 'Physical inactivity in leisure time and Inadequate consumption of fruits and vegetables', 'Saturated fat', 'Alcohol and Smoking' and 'Sedentary behaviour and Sugar', explaining 52·01 % of the total variance. Overall, greater adherence to 'Saturated fat' and 'Alcohol and Smoking' patterns was observed among men and those with lower education level. The 'Sedentary behaviour and Sugar' and 'Physical inactivity in leisure time and Inadequate consumption of fruits and vegetables' patterns had greater adherence among younger individuals, and the first was associated with higher education whereas the second with less education among individuals residing in the North and Northeast regions. CONCLUSIONS: Risk behaviour patterns for NCD were heterogeneous, reflecting the socio-economic and demographic differences in Brazil.

Overexpression of eukaryotic initiation factor 5A (eIF5A) affects susceptibility to benznidazole in Trypanosoma cruzi populations.

Eukaryotic initiation factor 5A (eIF5A) is a conserved protein with an essential role in translation elongation. Using one and two-dimensional western blotting, we showed that the eIF5A protein level was 2-fold lower in benznidazole (BZ)-resistant (BZR and 17LER) Trypanosoma cruzi populations than in their respective susceptible counterparts (BZS and 17WTS). To confirm the role of eIF5A in BZ resistance, we transfected BZS and 17WTS with the wild-type eIF5A or mutant eIF5A-S2A (in which serine 2 was replaced by alanine). Upon overexpressing eIF5A, both susceptible lines became approximately 3- and 5-fold more sensitive to BZ. In contrast, the eIF5A-S2A mutant did not alter its susceptibility to BZ. These data suggest that BZ resistance might arise from either decreasing the translation of proteins that require eIF5A, or as a consequence of differential levels of precursors for the hypusination reactions (e.g., spermidine and trypanothione), both of which alter BZ's effects in the parasite.

Molecular characterization of Cyclophilin (TcCyP19) in Trypanosoma cruzi populations susceptible and resistant to benznidazole.

Cyclophilin (CyP), a peptidyl-prolyl cis/trans isomerase, is a key molecule with diverse biological functions that include roles in molecular chaperoning, stress response, immune modulation, and signal transduction. In this respect, CyP could serve as a potential drug target in disease-causing parasites. Previous studies employing proteomics techniques have shown that the TcCyP19 isoform was more abundant in a benznidazole (BZ)-resistant Trypanosoma cruzi population than in its susceptible counterpart. In this study, TcCyP19 has been characterized in BZ-susceptible and BZ-resistant T. cruzi populations. Phylogenetic analysis revealed a clear dichotomy between Cyphophilin A (CyPA) sequences from trypanosomatids and mammals. Sequencing analysis revealed that the amino acid sequences of TcCyP19 were identical among the T. cruzi samples analyzed. Southern blot analysis showed that TcCyP19 is a single-copy gene, located in chromosomal bands varying in size from 0.68 to 2.2 Mb, depending on the strain of T. cruzi. Northern blot and qPCR indicated that the levels of TcCyP19 mRNA were twofold higher in drug-resistant T. cruzi populations than in their drug-susceptible counterparts. Similarly, as determined by two-dimensional gel electrophoresis immunoblot, the expression of TcCyP19 protein was increased to the same degree in BZ-resistant T. cruzi populations. No differences in TcCyP19 mRNA and protein expression levels were observed between the susceptible and the naturally resistant T. cruzi strains analyzed. Taken together, these data indicate that cyclophilin TcCyP19 expression is up-regulated at both transcriptional and translational levels in T. cruzi populations that were in vitro-induced and in vivo-selected for resistance to BZ.

Antistaphylococcal and biofilm inhibitory activities of gallic, caffeic, and chlorogenic acids.

Staphylococcus aureus is a Gram-positive pathogen which is able to form biofilms, exhibiting a more pronounced resistance to antibiotics and disinfectants. The hurdles posed in eradicating biofilms have driven the search for new compounds able to fight these structures. Phenolic compounds constitute one of the most numerous and ubiquitous group of plant secondary metabolites with many biological activities. The aim of the present work was to study the potential antimicrobial and antibiofilm properties of gallic, caffeic, and chlorogenic acids against S. aureus as well to elucidate its mechanism of action. It was concluded that the phenolic acids studied in this work have antistaphylococcal properties. For instance, gallic acid is able to influence the adhesion properties of S. aureus. The phenolic acids tested were also able to inhibit the production of α-hemolysin by this microorganism, with the exception of chlorogenic acid. Regarding its mechanism of action, caffeic acid interferes with the stability of the cell membrane and with the metabolic activity of the cells of S. aureus.

Stumps of Eucalyptus globulus as a source of antioxidant and antimicrobial polyphenols.

These past years have seen an enormous development of the area of natural antioxidants and antimicrobials. Eucalyptus globulus is widely cultivated in subtropical and Mediterranean regions in intensive short rotation coppice plantations. In the Portuguese context, E. globulus is the third species in terms of forest area. The stump is the basal part of the tree, including the near-the-ground stem portion and the woody roots that remain after stem felling. The purpose of this work was to study the phytochemical profile and to evaluate the antioxidant and antimicrobial properties of several crude stump wood and stump bark extracts of E. globulus, comparing it with similar extracts of E. globulus wood (industrial chips). The results showed the presence of high concentrations of total phenolic compounds (>200 mg GAE/g extract) and flavonoids (>10 mg QE/g extract) in E. globulus stump extracts. Generally the stump wood extracts stands out from the other ones, presenting the highest percentages of inhibition of linoleic acid oxidation. It was also possible to conclude that the extracts were more active against Gram-positive bacteria, presenting low MIC values. This study thus provides information supporting the economic valorization of E. globulus stump wood.