Potential of the Blue Calm® food supplement in the treatment of alcohol withdrawal-induced anxiety in adult zebrafish (Danio rerio).

Alcohol use disorder (AUD) is characterized by a set of behavioral, cognitive, nutritional, and physiological phenomena derived from the uncontrolled use of alcoholic beverages. There are cases in which AUD is associated with anxiety disorder, and when untreated, it requires careful pharmacotherapy. Blue Calm® (BC) is a food supplement indicated to aid restorative sleep, which has traces of medicinal plant extracts, as well as myo-inositol, magnesium bisglycinate, taurine, and L-tryptophan as its main chemical constituents. In this context, this study aimed to evaluate the potential of the BC in the treatment alcohol withdrawal-induced anxiety in adult zebrafish (aZF). Initially, BC was submitted to antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl radical. Subsequently, the aZF (n = 6/group) were treated with BC (0.1 or 1 or 10 mg/mL; 20 µL; p.o.), and the sedative effect and acute toxicity (96 h) were evaluated. Then, the anxiolytic-like effect and the possible GABAergic mechanism were analyzed through the Light & Dark Test. Finally, BC action was evaluated for treating alcohol withdrawal-induced anxiety in aZF. Molecular docking was performed to evaluate the interaction of the major chemical constituents of BC with the GABAA receptor. BC showed antioxidant potential, a sedative effect, was not toxic, and all doses of BC had an anxiolytic-like effect and showed potential for the treatment of alcohol withdrawal-induced anxiety in aZF. In addition to the anxiolytic action, the main chemical constituents of BC were confirmed in the molecular docking, thus suggesting that BC is an anxiolytic that modulates the GABAergic system and has pharmacological potential for the treatment of alcohol withdrawal-induced anxiety.

Antinociceptive, anti-inflammatory and hypoglycemic activities of the ethanolic Turnera subulata Sm. flower extract in adult zebrafish (Danio rerio).

Turnera subulata Sm. belongs to the family Turneraceae and is found in Brazil. The present study evaluated the antinociceptive, anti-inflammatory, and hypoglycemic potential of T. subulata flower extract (EtFloTsu) in zebrafish (Danio rerio). The total phenol and flavonoid contents of EtFloTsu were determined and identified using the Folin Ciocalteu reagent and aluminum chloride (AlCl3), respectively. The constituents of the extract were identified by HPLC-DAD, and the in vitro antioxidant activity (DPPH) was determined, toxicity in brine shrimp, and acute toxicity of 96 h in adult zebrafish. In addition, adult zebrafish (n = 6/fish) were treated orally with EtFloTsu (4, 20, or 40 mg/kg; vo) and subjected to formalin-induced nociception tests (with its possible mechanism of action with camphor), carrageenan-induced inflammation, and D-glucose-induced hyperglycemia (111 mM). Oxidative stress in the liver and brain tissues was assessed. EtFloTsu showed high levels of phenolic and flavonoid compounds with antioxidant activity. The phytochemicals chlorogenic acid, luteolin-7-o-glucoside, vitexin, and apigenin-7-o-glucoside were also identified in EtFloTsu. The synergism between these constituents was possibly responsible for the antinociceptive (via TRPA1), anti-inflammatory, and hypoglycemic effects of EtFloTsu in adult zebrafish, without causing toxicity in animals. Therefore, T. subulata flowers have therapeutic agents that could treat pain, inflammation, diabetes, and related complications.Communicated by Ramaswamy H. Sarma.

UPLC-MS-ESI-QTOF Analysis and Antifungal Activity of Aqueous Extracts of Spondias tuberosa.

This study aimed to identify the chemical composition of the Spondias tuberosa aqueous leaf and root extracts (EALST and EARST) and to evaluate their effect, comparatively, against opportunistic pathogenic fungi. Ultra-Performance Liquid Chromatography Coupled to a Quadrupole/Time of Flight System (UPLC-MS-ESI-QTOF) was employed for chemical analysis. Candida albicans and C. tropicalis standard strains and clinical isolates were used (CA INCQS 40006, CT INCQS 40042, CA URM 5974, and CT URM 4262). The 50% Inhibitory Concentration for the fungal population (IC50) was determined for both the intrinsic action of the extracts and the extract/fluconazole (FCZ) associations. The determination of the Minimum Fungicidal Concentration (MFC) and the verification of effects over fungal morphological transitions were performed by subculture in Petri dishes and humid chambers, respectively, both based on micro-dilution. UPLC-MS-ESI-QTOF analysis revealed the presence of phenolic and flavonoid compounds. The association of the extracts with fluconazole, resulted in IC50 values from 2.62 µg/mL to 308.96 µg/mL. The MFC of the extracts was ≥16,384 µg/mL for all tested strains, while fluconazole obtained an MFC of 8192 µg/mL against C. albicans strains. A reduction in MFC against CA URM 5974 (EALST: 2048 µg/mL and EARST: 1024 µg/mL) occurred in the extract/fluconazole association.

Antinociceptive activity of 3ß-6ß-16ß-trihydroxylup-20 (29)-ene triterpene isolated from Combretum leprosum leaves in adult zebrafish (Danio rerio).

Drugs used to treat pain are associated with adverse effects, increasing the search for new drugs as an alternative treatment for pain. Therefore, we evaluated the antinociceptive behavior and possible neuromodulation mechanisms of triterpene 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene (CLF-1) isolated from Combretum leprosum leaves in zebrafish. Zebrafish (n = 6/group) were pretreated with CLF-1 (0.1 or 0.3 or 1.0 mg/mL; i.p.) and underwent nociception behavior tests. The antinociceptive effect of CFL-1 was tested for modulation by opioid (naloxone), nitrergic (L-NAME), nitric oxide and guanylate cyclase synthesis inhibitor (methylene blue), NMDA (Ketamine), TRPV1 (ruthenium red), TRPA1 (camphor), or ASIC (amiloride) antagonists. The corneal antinociceptive effect of CFL-1 was tested for modulation by TRPV1 (capsazepine). The effect of CFL-1 on zebrafish locomotor behavior was evaluated with the open field test. The acute toxicity study was conducted. CLF-1 reduced nociceptive behavior and corneal in zebrafish without mortalities and without altering the animals' locomotion. Thus, CFL-1 presenting pharmacological potential for the treatment of acute pain and corneal pain, and this effect is modulated by the opioids, nitrergic system, NMDA receptors and TRP and ASIC channels.

Orally hypoglycemic activity of an insulin mimetic glycoprotein isolated from Cnidoscolus quercifolius Pohl. (Euphorbiaceae) seeds, Cq-IMP.

The genus Cnidoscolus (Euphorbiaceae) is widely distributed in tropical areas. In the Northeast of Brazil, the species C. quercifolius is endemic and has been used in traditional medicine. In this study, a novel protein was isolated from C. quercifolius seeds and characterized by its molecular weight, primary structure, isoelectric point (pI), and carbohydrate content. The hypoglycemic activity of this protein was investigated by in vitro assay with the RIN-5F glucose-responsive cell line and in vivo test using alloxan-induced diabetic mice models. In addition, safe use of the protein was also investigated by cytotoxicity, hemagglutinating, and immunogenicity assays. The protein which was named Cq-IMP (Cnidoscolus quercifolius - Insulin Mimetic Protein) showed a single 11.18 KDa glycopolypeptide chain (16.4% of carbohydrates, m/m), pI of 8.0 and N-terminal sequence (TKDPELKQcKKQQKKqQQYDDDDKK) with similarity around 46-62% to sucrose binding protein-like and vicilin-like protein that was confirmed by mass spectrometry tryptic peptides analysis. Besides that, Cq-IMP presented anti-insulin antibody cross-reactivity as hypoglycemic activity in both in vitro and in vivo models. Additionally, it did not present any toxicity by methods tested. In conclusion, Cq-IMP is an insulin-mimetic protein, with a potent hypoglycemic activity and no toxicity showing great potential for therapeutic applications and drug development.

Synthesis of Quercetin-Metal Complexes, In Vitro and In Silico Anticholinesterase and Antioxidant Evaluation, and In Vivo Toxicological and Anxiolitic Activities.

The level of acetylcholine, a neurotransmitter essential for processing memory and learning, is lower in the brains of patients with Alzheimer's disease due to the higher concentration of the enzyme acetylcholinesterase. The main compounds used for Alzheimer's treatment are acetylcholinesterase inhibitors. Quercetin coordination complexes with the metal ions Cu+2, Zn+2, Ni+2, Co+2, and Fe+2 were synthesized to investigate their potential use against Alzheimer's disease, by evaluating the inhibition of acetylcholinesterase in vitro and in silico, as well as the antioxidant activity, toxicity, and anxiolytic action in the zebrafish (Danio rerio) model. The organic complexes were characterized by UV-Vis and FT-IR. The spectral information suggested that coordination of metals occurs with the carbonyl group and OH linked to the C-3 carbon of quercetin. The quercetin-Fe (QFe) complex presented the best antioxidant and antiacetylcholinesterase actions, and these results were confirmed by molecular docking. In the toxicity and locomotor evaluation, the quercetin molecules and the synthesized complexes, mainly QCu and QZn derivatives, showed the highest degree of inhibition of the fish's locomotor activity, suggesting a possible anxiolytic action. Then, quercetin complexes with metals, mainly with Fe+2, represent valuable compounds and deserve more investigation as promising agents against Alzheimer's disease.

Metabolic profile of pitaya (Hylocereus polyrhizus (F.A.C. Weber) Britton & Rose) by UPLC-QTOF-MSE and assessment of its toxicity and anxiolytic-like effect in adult zebrafish.

Pitaya is a Cactacea with potential for economic exploitation, due to its high commercial value and its functional components - such betalains, oligosaccharides and phenolic compounds. Although the biological activities of pitaya have been studied using in vivo and in vitro models (anti-inflammatory and antiproliferative activities, as example), its anxiolytic-like effect is still unexplored. Therefore, the aim of this work was to perform a characterization of pulp and peel of pitaya (Hylocereus polyrhizus (F.A.C. Weber) Britton & Rose) using UPLC-QTOF-MSE, and to assess its toxicity and anxiolytic-like effect in adult zebrafish (Danio rerio). The results showed 16 and 15 compounds (in pulp and peel, respectively), including maltotriose, quercetin-3-O-hexoside, and betalains, putatively identified by UPLC-QTOF-MSE. Thus, pitaya pulp and peel showed no toxicity in both models tested (Vero cell lines and zebrafish model, LC50 ˃ 1 mg/mL); and a significant anxiolytic activity, since the treated fish reduced the permanence in the clear zone (Light & Dark Test) compared to that in the control, exhibiting anxiolytic-simile effect of diazepam. However, these effects were reduced by pre-treatment with the flumazenil suggesting that the pulp and peel of pitaya are anxiolytics agents mediated via the GABAergic system. These findings suggested that H. polyrizhus has the potential of developing an alternative plant-derived anxiolytic therapy. In addition, pitaya peel (which is a waste in the food industry) should be regarded as a valuable product, which has the potential as an economic value-added ingredient for anxiety disorders.

Ethnobotanic, phytochemical uses and ethnopharmacological profile of genus Cnidoscolus spp. (Euphorbiaceae): A comprehensive overview.

The application of medicinal plants are the most important biotechnological alternative in the treatment of numerous diseases, especially in developing countries, such as Brazil. Among them, we specified some specimens of the genus Cnidoscolus used as phytotherapies, with healing properties, analgesic, anti-inflammatory, antibiotic and diuretic, anticancer, among others. Such effects are possibly associated with the presence of terpenoids, alkaloids, coumarins, flavonoids phenolic compounds, among others. Thus, the objective of this work was to evaluate in the literature the studies on the phytochemical, ethnopharmacological and biotechnological applications of this genus, from 1998 to 2017. Among the sixty-one studies reported in this review, ten species are popularly utilized to pharmacological and/or biotechnological applications. Cnidoscolus aconitifolius and Cnidoscolus chayamansa are the most cited species, which were also supported by either animal or cellular investigations indicating some beneficial pharmacological actions like antioxidant, anti-inflammatory and potential cytotoxic activity. The plant parts of this genus under study are important as sources for the isolation and identification of bioactive molecules with biotechnological applications, among the many diseases treated with this phytotherapy. Given these verdicts, ethnopharmacological approaches are significant systematic tools in the determination of plant species that exhibit medicinal and nutritional purposes. The results presented here should further stimulate the development of validation studies to ensure the safe and effective use of these plant species.