Vanilla from Brazilian Atlantic Forest: In vitro and in silico toxicity assessment and high-resolution metabolomic analysis of Vanilla spp. ethanolic extracts.

The natural vanilla market, which generates millions annually, is predominantly dependent on Vanilla planifolia, a species characterized by low genetic variability and susceptibility to pathogens. There is an increasing demand for natural vanilla, prized for its complex, authentic, and superior quality compared to artificial counterparts. Therefore, there is a necessity for innovative production alternatives to ensure a consistent and stable supply of vanilla flavors. In this context, vanilla crop wild relatives (WRs) emerge as promising natural sources of the spice. However, these novel species must undergo toxicity assessments to evaluate potential risks and ensure safety for consumption. This study aimed to assess the non-mutagenic and non-carcinogenic properties of ethanolic extracts from V. bahiana, V. chamissonis, V. cribbiana, and V. planifolia through integrated metabolomic profiling, in vitro toxicity assays, and in silico analyses. The integrated approach of metabolomics, in vitro assays, and in silico analyses has highlighted the need for further safety assessments of Vanilla cribbiana ethanolic extract. While the extracts of V. bahiana, V. chamissonis, and V. planifolia generally demonstrated non-mutagenic properties in the Ames assay, V. cribbiana exhibited mutagenicity at high concentrations (5000 µg/plate) in the TA98 strain without metabolic activation. This finding, coupled with the dose-dependent cytotoxicity observed in WST-1 (Water Soluble Tetrazolium) assays, a colorimetric method that assesses the viability of cells exposed to a test substance, underscores the importance of concentration in the safety evaluation of these extracts. Kaempferol and pyrogallol, identified with higher intensity in V. cribbiana, are potential candidates for in vitro mutagenicity. Although the results are not conclusive, they suggest the safety of these extracts at low concentrations. This study emphasizes the value of an integrated approach in providing a nuanced understanding of the safety profiles of natural products, advocating for cautious use and further research into V. cribbiana mutagenicity.

Anti-Sporothrix Activity of Novel Copper-Itraconazole Complexes.

A series of new metal complexes, [Cu(ITZ)2Cl2] ⋅ 5H2O (1), [Cu(NO3)2(ITZ)2] ⋅ 3H2O ⋅ C4H10O (2) and [Cu(ITZ)2)(PPh3)2]NO3 ⋅ 5H2O (3) were synthesized by a reaction of itraconazole (ITZ) with the respective copper salts under reflux. The metal complexes were characterized by elemental analyses, molar conductivity, 1H and 13C{1H} nuclear magnetic resonance, UV-Vis, infrared and EPR spectroscopies. The antifungal activity of these metal complexes was evaluated against the main sporotrichosis agents: Sporothrix brasiliensis, Sporothrix schenkii, and Sporothrix globosa. All three new compounds inhibited the growth of S. brasiliensis and S. schenckii at lower concentrations than the free azole, with complex 2 able to kill all species at 4 µM and induce more pronounced alterations in fungal cells. Complexes 2 and 3 exhibited higher selectivity and no mutagenic effect at the concentration that inhibited fungal growth and affected fungal cells. The strategy of coordinating itraconazole (ITZ) to copper was successful, since the corresponding metal complexes were more effective than the parent drug. Particularly, the promising antifungal activity of the Cu-ITZ complexes makes them potential candidates for the development of an alternative drug to treat mycoses.

In vitro chemopreventive and cytotoxic effects of Amazon mosses Leucobryum martianum (Hornsch.) and Leucobryum laevifolium (Broth) extracts.

Several bioactive compounds, such as polyphenols, demonstrate low toxicity and prominent effects on cancer cells with antioxidant, anti-inflammatory, and antitumor activities. Such compounds can be found in Amazon mosses Leucobryum martianum (Hornsch.) Hampe ex Müll. Hal. (Hornsch.) and Leucobryum laevifolium (Broth). Antimutagenic assay with Salmonella enterica serovar Typhimurium and cytotoxicity with different eukaryotic cell lines were carried out to screen aqueous, hydroalcoholic, and ethanolic extracts of those Amazon mosses for anticancer potential. The results indicate the capacity of all extracts of both mosses to exert chemopreventive effects against 4-nitroquinoline-N-oxide (4NQO) and 2-aminoanthracene (2-AA), which are direct or indirect mutagens. In particular, the ethanolic and aqueous extract from L. martianum. The ethanolic extract from L. martianum induces significant cytotoxicity by mitochondrial metabolism and cell membrane disruption pathways to tumor or non-tumor cells. The aqueous extract from L. martianum showed a mainly cytotoxic response in the HepG2 cells, a human liver carcinoma, reaching ~90% cytotoxicity. The same extract did not induce significant damage to normal liver cells (F C3H cells) by membrane interaction pathway. The selective cytotoxicity in the aqueous extract of L. martianum makes it a candidate against liver cancer. Further studies, including in vivo models, are necessary to validate the efficacy and safety of the aqueous extract of L. martianum.

Echinodorus macrophyllus: Acute toxicological evaluation of hydroxycinnamoyl derivatives from SF1 subfractions.

ETHNOPHARMACOLOGICAL RELEVANCE: Echinodorus macrophyllus (Kunth.) Micheli (Alismataceae), known as chapéu-de-couro in Brazil, is popularly used to treat inflammatory diseases. We have previously demonstrated a significant reduction in the acute inflammation for the aqueous extract of E. macrophyllus (AEEm) and its ethanolic fraction (Fr20) and described that hydroxycinnamoyl derivatives present in SF1 (Fr20 subfraction) showed higher anti-inflammatory properties by mechanisms that include a reduction of TNF-α, IL-1ß, CKCL1/KC, LTB4, and PGE2 levels in exudate. AIM OF THE STUDY: This work describes the acute toxicological effect of SF1 subfraction on SW mice treated orally for five days in the air pouch model by evaluating the hematological and biochemical determinations on the blood samples; the relative organ weight and its histopathological analysis; the liver genotoxicity assessment and the activity of liver enzymes from xenobiotic metabolism. MATERIALS AND METHODS: Fr20 was earlier fractionated on the Sephadex LH-20 column, yielding mainly four subfractions, including SF1. The SF1 toxicity was evaluated in mice challenged with carrageenan on the air pouch inflammation model and orally treated for five days. The body weight was monitored daily, and the organs were weighed after the euthanasia. Hematological and biochemical determinations were carried out using specific commercial kits and following the protocols provided by the manufacturers. The organs were fixed, sectioned, processed for hematoxylin and eosin staining, and analyzed by light microscopy. Genotoxicity assessment was performed by the alkaline single-cell gel electrophoresis. Livers were processed for ethoxyresorufin-O-deethylase (EROD) and Glutathione S-transferase (GST) assays. RESULTS: SF1 exhibited low toxicity, as no significant discrepancy was observed in the relative weight of the body organs of mice. Moreover, the daily treatment with SF1 did not alter the number and percentage of red blood cells or hemoglobin concentration in the blood. The treatment with SF1 did not affect the creatinine concentration, but the 25 mg/kg dose reduced the plasma urea level and uric acid, suggesting its use in treating acute renal failure. The parameters analyzed did not present biochemical alterations indicative of liver disease. Regarding serum triglyceride and cholesterol levels, a significant decrease was detected in both parameters in mice treated with SF1. In addition, the histopathological analysis showed that inflammatory focus in the livers seemed more relevant in the control groups than in those treated. There were no significant changes in the renal or splenic tissues of animals treated with SF1. Treatment with SF1 also does not have a genotoxic effect on liver cells. CONCLUSION: Treatment with SF1 showed no toxicity in mice at doses equivalent to those recommended for humans, which provides evidence of the safety of the therapeutic use of this subfraction.

Marketable 1,3-dimethylamylamine and caffeine-based thermogenic supplements: Regulatory genotoxicity assessment through in vitro and in silico approaches.

The consumption of dietary supplements to enhance physical performance has increased significantly in the last century, especially thermogenic pre-workout supplements. Nevertheless, this industry has faced criticism for inadequate safety measures surveillance in regulatory issues regarding their products. The aims of our study were to investigate two pre-workout supplements with respect to (1) mutagenicity utilizing Salmonella/microsome assay; (2) genotoxicity employing cytokinesis-block micronucleus (CBMN) assay protocols; and (3) hepatocytoxicity using WST cell proliferation, activities of lactate dehydrogenase (LDH) and alkaline phosphatase using human liver carcinoma (HepG2) and mouse fibroblast (F C3H) cells. Oxidative stress was determined through glutathione (GSH) measurement and in silico for predictions of pharmacokinetics and toxicity for the most abundant isolated substances present in these supplements. Both supplements induced mutagenicity in all examined bacterial strains, especially in the presence of exogenous metabolism. Further, tested supplements significantly elevated the formation of micronuclei (MN) as well as other cellular phenomena. Concentration- and time-dependent curves were observed for hepatotoxicity in both studied cell lines. In addition, both supplements decreased levels of intracellular and extracellular GSH. In silico predictions showed that the isolated individual compounds failed to induce the observed outcomes. Our findings provide contributions to the molecular mechanisms underlying two pre-workout supplement-induced toxicity and the need for surveillance.

Anti-Trypanosoma cruzi Activity, Mutagenicity, Hepatocytotoxicity and Nitroreductase Enzyme Evaluation of 3-Nitrotriazole, 2-Nitroimidazole and Triazole Derivatives.

Chagas disease (CD), which is caused by Trypanosoma cruzi and was discovered more than 100 years ago, remains the leading cause of death from parasitic diseases in the Americas. As a curative treatment is only available for the acute phase of CD, the search for new therapeutic options is urgent. In this study, nitroazole and azole compounds were synthesized and underwent molecular modeling, anti-T. cruzi evaluations and nitroreductase enzymatic assays. The compounds were designed as possible inhibitors of ergosterol biosynthesis and/or as substrates of nitroreductase enzymes. The in vitro evaluation against T. cruzi clearly showed that nitrotriazole compounds are significantly more potent than nitroimidazoles and triazoles. When their carbonyls were reduced to hydroxyl groups, the compounds showed a significant increase in activity. In addition, these substances showed potential for action via nitroreductase activation, as the substances were metabolized at higher rates than benznidazole (BZN), a reference drug against CD. Among the compounds, 1-(2,4-difluorophenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanol (8) is the most potent and selective of the series, with an IC50 of 0.39 µM and selectivity index of 3077; compared to BZN, 8 is 4-fold more potent and 2-fold more selective. Moreover, this compound was not mutagenic at any of the concentrations evaluated, exhibited a favorable in silico ADMET profile and showed a low potential for hepatotoxicity, as evidenced by the high values of CC50 in HepG2 cells. Furthermore, compared to BZN, derivative 8 showed a higher rate of conversion by nitroreductase and was metabolized three times more quickly when both compounds were tested at a concentration of 50 µM. The results obtained by the enzymatic evaluation and molecular docking studies suggest that, as planned, nitroazole derivatives may utilize the nitroreductase metabolism pathway as their main mechanism of action against Trypanosoma cruzi. In summary, we have successfully identified and characterized new nitrotriazole analogs, demonstrating their potential as promising candidates for the development of Chagas disease drug candidates that function via nitroreductase activation, are considerably selective and show no mutagenic potential.

Antiproliferative and Apoptosis Effects of Hybrid Varieties of Vitis vinifera L. Sweet Sapphire and Sweet Surprise on Human Prostate Cancer Cells Using In Vitro and In Silico Approaches.

OBJECTIVE: Grape hybrids are characterized by different chemical compositions; often with high hybrids are characterized by different chemical compositions, often with a high phenolic content and a specific profile of anthocyanins. The aim of study was to characterize the constituents of hybrid Vitis vinifera L. varieties Sweet Sapphire (SA) and Sweet Surprise (SU) extracts and their influence on apoptosis induction and antiproliferative effects on human prostate cancer cells. METHODS: We used the MTT assay to evaluate the cytotoxic effect of extracts of SA and SU, on the prostate adenocarcinoma cell lines PC-3 and DU-145. To analyze the inhibiting impact by flow cytometry, used 24 and 48 hours. Anthocyanins were quantified by liquid chromatography and analysed by their absorption rate, hepatotoxicity, blood concentration, blood-brain barrier passage ability and maximum recommended dose by in silico approaches. RESULTS: Our results showed that malvidin derivatives present the highest content in both cultivars. We identified 14.46mg/100g malvidin-3-O-glycoside in SA and 2.76 mg/100 g in SU. A reduction in cell viability of DU-145 (45 and 65%) and PC-3 (63 and 67%) cells after 48h treatment with SA and SU, respectively, was found via MTT assay. Flow cytometry showed that the treatment with extracts from SA and SU had an inhibitory impact on cell development due to G2/M arrest and caused a rise in apoptotic cells compared to control group. None of the anthocyanin presented hepatotoxicity as well as blood-brain barrier passage ability. Peonidin 3-O-glucoside had the lower maximum recommended dose as well as the highest intestinal absorption rate. However, delphinidin 3-O-glucoside had the highest blood concentration values. CONCLUSION: The findings of this study highlight the potential of hybrid Vitis vinifera L. varieties as an important source of natural antioxidants and their protective effect against prostate cancer cells as well as elucidate in part their anthocyanin's metabolism.

Antiplasmodial, Trypanocidal, and Genotoxicity In Vitro Assessment of New Hybrid α,α-Difluorophenylacetamide-statin Derivatives.

BACKGROUND: Statins present a plethora of pleiotropic effects including anti-inflammatory and antimicrobial responses. A,α-difluorophenylacetamides, analogs of diclofenac, are potent pre-clinical anti-inflammatory non-steroidal drugs. Molecular hybridization based on the combination of pharmacophoric moieties has emerged as a strategy for the development of new candidates aiming to obtain multitarget ligands. METHODS: Considering the anti-inflammatory activity of phenylacetamides and the potential microbicidal action of statins against obligate intracellular parasites, the objective of this work was to synthesize eight new hybrid compounds of α,α-difluorophenylacetamides with the moiety of statins and assess their phenotypic activity against in vitro models of Plasmodium falciparum and Trypanosoma cruzi infection besides exploring their genotoxicity safety profile. RESULTS: None of the sodium salt compounds presented antiparasitic activity and two acetated compounds displayed mild anti-P. falciparum effect. Against T. cruzi, the acetate halogenated hybrids showed moderate effect against both parasite forms relevant for human infection. Despite the considerable trypanosomicidal activity, the brominated compound revealed a genotoxic profile impairing future in vivo testing. CONCLUSIONS: However, the chlorinated derivative was the most promising compound with chemical and biological profitable characteristics, without presenting genotoxicity in vitro, being eligible for further in vivo experiments.

In silico and in vitro assessment of anti-Trypanosoma cruzi efficacy, genotoxicity and pharmacokinetics of pentasubstituted pyrrolic Atorvastatin-aminoquinoline hybrid compounds.

Atorvastatin (AVA) is a third-generation statin with several pleiotropic effects, considered the last synthetic pharmaceutical blockbuster. Recently, our group described the effects of AVA on DNA damage prevention and against Trypanosoma cruzi infection. In this study, our aim was to evaluate the efficacy, safety, and in silico pharmacokinetic profile of four hybrids of aminoquinolines with AVA 4a-d against T. cruzi using in vitro and in silico models. These synthetic compounds were designed by hybridization of the pentapyrrolic moiety of AVA with the aminoquinolinic unit of chloroquine or primaquine. Pharmacokinetics (ADME) and toxicity parameters were predicted by SwissADME, admetSAR and LAZAR in silico algorithms. The trypanocidal activity of AVA-quinoline hybrids were evaluated in vitro against amastigotes and trypomastigotes of T. cruzi, from Y (Tc II) and Tulahuen (Tc VI) strains. In vitro cardiocytotoxicity was assessed using primary cultures of mouse embryonic cardiac cells and in vitro hepatocytotoxicity on bidimensional and 3D-cultured HepG2 cells. Genotoxicity was evaluated by Ames test and micronucleus assay. Despite the overall good in silico ADMET profile, all tested compounds were predicted to be hepatotoxic. All hybrid derivatives presented high trypanocidal activity, against both trypomastigote and intracellular forms of T. cruzi, presenting EC50's lower than 1 µM besides superior selectivity than the reference drug, without evidences of cardiotoxicity in vitro. The compounds 4a and 4b presented a time-dependent toxicity in monolayer culture of HepG2 but no detectable toxic effects in their spheroids, opposing to the in silico prediction. We can conclude that the AVA-aminoquinoline hybrids presented a hit profile as antiparasitic agents in synthetic pharmaceutical innovation platforms.

Antioxidant Capacity, Antitumor Activity and Metabolomic Profile of a Beetroot Peel Flour.

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.

In vitro genotoxicity assessment of graphene quantum dots nanoparticles: A metabolism-dependent response.

Nanomaterials are progressively being applied in different areas, including biomedical uses. Carbon nanomaterials are relevant for biomedical sciences because of their biocompatibility properties. Graphene quantum dots (GQD) have a substantial potential in drug-delivery nanostructured biosystems, but there is still a lack of toxicological information regarding their effects on human health and the environment. We thus evaluated the mutagenicity, cytotoxicity and genotoxicity of this nanomaterial using alternative methods applied in regulatory toxicology guidelines. The Ames test was carried out in the presence and absence of exogenous metabolization. Salmonella enterica serovar Typhimurium strains TA97a, TA98, TA100, TA102, TA104, and TA1535 were exposed to GQD with concentrations ranging from 1 to 1000 µg/plate. The mammal cell viability assays were carried out with HepG2 and 3T3BalbC cell lineages and the in vitro Cytokinesis-Block Micronucleus assay (CBMN) was applied for 24 h of exposure in non-cytotoxic concentrations. Mutagenicity was induced in the TA97a strain in the absence of exogenous metabolization, but not in its presence. Mutagenicity was also detected in the TA102 strain in the assay with exogenous metabolization, suggesting redox misbalance mutagenicity. The WST-1 and LDH assays demonstrated that GQD decreased cell viability, especially in 3T3BalbC cells, which showed more sensitivity to the nanomaterial. GQD also increased micronuclei formation in 3T3BalbC and caused a cytostatic effect. No significant impact on HepG2 micronuclei formation was observed. Different metabolic systems interfered with the mutagenic, cytotoxic, and genotoxic effects of GQD, indicating that liver metabolism has a central role in the detoxification of this nanomaterial.

Toxicogenetic assessment of a pre-workout supplement: In vitro mutagenicity, cytotoxicity, genotoxicity and glutathione determination in liver cell lines and in silico ADMET approaches.

The benefits of practicing physical activity, such as weight loss and control, are commonly associated with caloric restriction diets and may be improved by the ingestion of thermogenic and ergogenic supplements. However, there is a lack of safety data on commonly marketed nutritional supplements. Therefore, this investigation aims to evaluate a pre-workout supplement for mutagenicity using the Ames test, hepatocytoxicity in HepG2 and F C3H cells after 24 h, 48 h and 72 h, genotoxicity using the CBMN assay, determination of gluthatione activity and computational prediction of the three major isolated compounds present in the supplement. The mutagenicity test showed a mutagenic response in TA98 His+ revertants of 5 mg/plate in the presence of metabolic activation, cytotoxicity in TA98 of 5 mg/plate in the absence of metabolic conditions, and in TA102 of 0.5 mg/plate both in the presence and absence of metabolic activation. In our in vitro eukaryotic cell viability, WST-1, LDH and alkaline phosphatase assays, the supplement showed hepatocytotoxicity both dose-dependently and time-dependently. In the cytokinesis blocking micronuclei assay, the supplement induced micronuclei, nuclear buds, nucleoplasmatic, bridge formation, and a decreased in nuclear division. In addition, the supplement decreased intra and extracellular GSH. Computational analysis showed that the three isolated compounds most present in the supplement have the potential to cause hepatotoxicity. In the present investigation, the pre-workout supplement induced mutagenic, genotoxic, and cytotoxic responses and GSH decrease. Thus, considering food safety and public health sanitary vigilance, the consumption of this pre-workout supplement may harm the health of its consumers.

A novel naphthoquinone derivative shows selective antifungal activity against Sporothrix yeasts and biofilms.

Sporotrichosis is a subcutaneous mycosis that affects humans and animals, with few therapeutic options available in the pharmaceutical market. We screened the in vitro antifungal activity of fourteen 1,4-naphthoquinones derivative compounds against Sporothrix brasiliensis and Sporothrix schenckii, the main etiological agents of sporotrichosis in Latin America. The most active compound was selected for further studies exploring its antibiofilm activity, effects on yeast morphophysiology, interaction with itraconazole, and selectivity to fungal cells. Among the fourteen 1,4-naphthoquinones tested, naphthoquinone 5, a silver salt of lawsone, was the most active compound. Naphthoquinone 5 was able to inhibit Sporothrix biofilms and induced ROS accumulation, mitochondrial disturbances, and severe plasmatic membrane damage in fungal cells. Furthermore, naphthoquinone 5 was ten times more selective towards fungal cells than fibroblast, and the combination of itraconazole with naphthoquinone 5 improved the inhibitory activity of the azole. Combined, the data presented here indicate that the silver salt naphthoquinone 5 exerts promising in vitro activity against the two main agents of sporotrichosis with important antibiofilm activity and a good toxicity profile, suggesting it is a promising molecule for the development of a new family of antifungals.

Plinia cauliflora (Mart.) Kausel (Jaboticaba) leaf extract: In vitro anti-Trypanosoma cruzi activity, toxicity assessment and phenolic-targeted UPLC-MSE metabolomic analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Plinia cauliflora (Mart.) Kausel, known as Brazilian grape or jaboticaba, is widely used in Brazilian traditional medicine to treat infectious and inflammatory disorders. However, several aspects of its biological potential remain unclear, such as toxicity and effects on pathogenic protozoa. AIM OF THE STUDY: Investigate the phenolic composition, the in vitro and in silico toxicity profile, and the anti-Trypanosoma cruzi activity of the phenolics-enriched hydromethanolic extract of P. cauliflora leaf. MATERIAL AND METHODS: Phytochemical analysis was performed ultra-performance liquid chromatography-mass spectrometry (UPLC-MSE). Mutagenicity, genotoxicity and eukaryotic cytotoxicity was evaluated by Ames test, cytokinesis-block micronucleus and colorimetric assays, respectively, alongside with a computational prediction of the major compound's pharmacokinetics and toxicity. Anti-T. cruzi activity was investigated on T. cruzi bloodstream trypomastigotes. RESULTS: A total of 14 phenolic compounds were identified, including 11 flavonoids and 2 phenolic acids. No positive response regarding mutagenic potential was detected in Salmonella strains TA97, TA98, TA100, TA102, TA104, both in absence or presence of metabolic activation. The extract induced significant dose-response reduction on nuclear division indexes of HepG2 cells, suggesting cytostatic effects, with no micronuclei induction on cytokinesis-block micronucleus assay. Likewise, it also presented cytotoxic effects, inducing HepG2 and F C3H dose and time dependently cell death through cell membrane damage and more evidently by mitochondrial dysfunction. A dose-response curve of in vitro trypanocidal activity was observed against T. cruzi bloodstream trypomastigotes after 2 and 24 h of exposure. In silico predictions of most abundant compounds' structural alerts, pharmacokinetics and toxicity profile indicates a moderately feasible druglikeness profile and low toxicity for them, which is compatible with in vitro results. CONCLUSIONS: The present study demonstrated that P. cauliflora leaf extract is a potential source of antiparasitic bioactive compounds, however it presents cytotoxic effects in liver cell lines.

In vitro biochemical characterization and genotoxicity assessment of Sapindus saponaria seed extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Sapindus saponaria, also popularly known as soapberry, has been used in folk medicinal values because of its therapeutic properties and several compounds in its composition, which represent a target in potential for drug discovery. However, few data about its potential toxicity has been reported. AIM OF THE STUDY: Plant proteins can perform essential roles in survival, acting as defense mechanism, as well functioning as important molecular reserves for its natural metabolism. The aim of the current study was to investigate the in vitro toxicity profile of protein extract of S. saponaria and detect protein potentially involved in biological effects such as collagen hydrolysis and inhibition of viral proteases. MATERIALS AND METHODS: Protein extract of soapberry seeds was investigated for its cytotoxic and genotoxic action using the Ames test. The protein extract was also subjected to a partial purification process of a protease and a protease inhibitor by gel chromatography filtration techniques and the partially isolated proteins were characterized biochemically. RESULTS: Seed proteins extract of S. saponaria was evaluated until 100 µg/mL concentration, presenting cytotoxicity and mutagenicity in bacterial model mostly when exposed to exogenous metabolic system and causing cytotoxic and genotoxic effects in HepG2 cells. The purification and partial characterization of a serine protease (43 kDa) and a cysteine protease inhibitor (32.8 kDa) from protein extract of S. Saponaria, corroborate the idea of ​​the biological use of the plant as an insecticide and larvicide. Although it shows cytotoxic, mutagenic and genotoxic effects. CONCLUSION: The overall results of the present study provide supportive data on the potential use of proteins produced in S. saponaria seeds as pharmacological and biotechnological agents that can be further explored for the development of new drugs.

Metabolomic analysis of Cyrtopodium glutiniferum extract by UHPLC-MS/MS and in vitro antiproliferative and genotoxicity assessment.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of orchids have been traditionally used as human phytotherapeutics. Cyrtopodium flavum, for example, due to the analgesic and anti-inflammatory properties, beside the capacity of heal skin lesions has been focus of research. Also Cyrtopodium glutiniferum, an orchid found in the Brazilian southeastern rainforest, is known to synthesize anti-inflammatory glucomannans in the pseudobulbs, as other potentially therapeutic compounds. AIM OF THE STUDY: We have reported the first metabolomic analysis focused on the phenols expression of the neotropical orchid Cyrtopodium glutiniferum Raddi, besides free radical scavenging, anti-inflammatory and antiproliferative activities, and the genotoxicity properties of the aqueous extract. MATERIAL AND METHODS: The metabolomics of C. glutiniferum aqueous extract was performed through UHPLC-MSn acquisition. We have detected the scavenging potential of the extract using DPPH assay. The genotoxic potential was performed by Ames Test (0-5000 µg mL-1) and micronucleous assay (0-5000 µg mL-1) in RAW264.7 cells. The cytotoxic potential of the extract against RAW264.7 was tested by WST-1 assay (0-500 µg mL-1). And after all, the RAW264.7 cells were treated with non-cytotoxic concentrations of C. glutiniferum (0-50 µg mL-1) to evaluate the antiproliferative and anti-inflammatory potential, besides the mitochondrial activity. RESULTS: From the 55 molecules identified, 45.5% belonged to the phenolic compounds database from Phenol Explorer, 29% to an in-house Orchidaceae molecules database, and 25.5% to both. Among the identified phenolic compounds, 18 subclasses were discriminated, being phenanthrenes the most abundant. Doses-dependent of C. glutiniferum extracts were able to induce DPPH free radicals scavenging and also to increase TA100 His+ revertants, in metabolic environment, showing mutagenicity just in the highest concentration, of 5 mg/plate. On Eukaryotic cell models, the extract also has induced dose-response and time-response cytotoxicity against RAW264.7 macrophages, mainly after 48 h and 72 h, even though the extract has not been able to induce the increase of micronucleated cells and mitotic index alteration on Micronucleus assay. The activation and proliferation of macrophages cultures were downregulated after 24 h and 48 h by the non-cytotoxic concentrations of the extract in a dose-dependent manner. CONCLUSIONS: The Cyrtopodium glutiniferum metabolomics, anti-inflammatory and anti-proliferative properties observed in this study suggest a therapeutic efficacy of the orchid extract applied in folk medicine.

Graphene quantum dots unraveling: Green synthesis, characterization, radiolabeling with 99mTc, in vivo behavior and mutagenicity.

Graphene is one of the crystalline forms of carbon, along with diamond, graphite, carbon nanotubes, and fullerenes, and is considered as a revolutionary and innovating product. The use of a graphene-based nanolabels is one of the latest and most prominent application of graphene, especially in the field of diagnosis and, recently, in loco radiotherapy when coupled with radioisotopes. However, its biological behavior and mutagenicity in different cell or animal models, as well as the in vivo functional activities, are still unrevealed. In this study we have developed by a green route of synthesizing graphene quantum dots (GQDs) and characterized them. We have also developed a methodology for direct radiolabeling of GQDs with radioisotopes.Finally; we have evaluated in vivo biological behavior of GQDs using two different mice models and tested in vitro mutagenicity of GQDs. The results have shown that GQDs were formed with a size range of 160-280 nm, which was confirmed by DRX and Raman spectroscopy analysis, corroborating that the green synthesis is an alternative, environmentally friendly way to produce graphene. The radiolabeling test has shown that stable radiolabeled GQDs can be produced with a high yield (>90%). The in vivo test has demonstrated a ubiquitous behavior when administered to healthy animals, with a high uptake by liver (>26%) and small intestine (>25%). Otherwise, in an inflammation/VEGF hyperexpression animal model (endometriosis), a very peculiar behavior of GQDs was observed, with a high uptake by kidneys (over 85%). The mutagenicity test has demonstrated A:T to G:C substitutions suggesting that GQDs exhibits mutagenic activity.

Cytotoxicity, genotoxicity, transplacental transfer and tissue disposition in pregnant rats mediated by nanoparticles: the case of magnetic core mesoporous silica nanoparticles.

Whether in the cosmetic or as therapeutic, the use of nanoparticles has been increasing and taking on global proportion. However, there are few studies about the physical potential of long-term use or use in special conditions such as chronic, AIDS, pregnant women and other special health circumstances. In this context, the study of the mutagenicity and the transplacental passage represents an important and reliable model for the primary evaluation of potential health risks, especially maternal and child health. In this study we performed mutagenicity, cytotoxic and transplacental evaluation of magnetic core mesoporous silica nanoparticles, radiolabeled with 99mTc for determination of toxicogenic and embryonic/fetuses potential risk in animal model. Magnetic core mesoporous silica nanoparticles were produced and characterized by obtaining nanoparticles with a size of (58.9 ± 8.1 nm) in spherical shape and with intact magnetic core. The 99 m Tc radiolabeling process demonstrated high efficacy and stability in 98% yield over a period of 8 hours of stability. Mutagenicity assays were performed using Salmonella enteric serovar Typhimurium standard strains TA98, TA100 and TA102. Cytotoxicity assays were performed using WST-1. The transplacental evaluation assays were performed using the in vivo model with rats in two periods: embryonic and fetal stage. The results of both analyzes corroborate that the nanoparticles can i) generate DNA damage; ii) generate cytotoxic potential and iii) cross the transplantation barrier in both stages and bioaccumulates in both embryos and fetuses. The results suggest that complementary evaluations should be conducted in order to attest safety, efficacy and quality of nanoparticles before unrestricted approval of their use.

New hydrazides derivatives of isoniazid against Mycobacterium tuberculosis: Higher potency and lower hepatocytotoxicity.

Tuberculosis (TB) is one of the leading causes of death worldwide. The emergence of multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) and TB-HIV co-infection are major public health challenges. The anti-TB drugs of first choice were developed more than 4 decades ago and present several adverse effects, making the treatment of TB even more complicated and the development of new chemotherapeutics for this disease imperative. In this work, we synthesized two series of new acylhydrazides and evaluated their activity against different strains of Mtb. Derivatives of isoniazid (INH) showed important anti-Mtb activity, some being more potent than all anti-TB drugs of first choice. Moreover, three compounds proved to be more potent than INH against resistant Mtb. The Ames test showed favorable results for two of these substances compared to INH, one of which presented expressly lower toxicity to HepG2 cells than that of INH. This result shows that this compound has the potential to overcome one of the main adverse effects of this drug.

The impact of maternal anti-retroviral therapy on cytokine profile in the uninfected neonates.

The number of HIV-infected young women has been increasing since the beginning of the AIDS epidemic. The objective of the present study was to investigate the impact of anti-retroviral treatment (ART) of HIV-1-infected pregnant women (PW) on cytokine profile of uninfected neonates. Our results demonstrated that higher levels of IL-1ß and TNF-α associated with lower IL-10 production were detected in the plasma obtained from neonates born from ART-treated PW. Furthermore, the production of TNF- α and IFN-γ was also significantly higher in polyclonally-activated T cells from those neonates. This elevated pro-inflammatory pattern detected by these activated-T cells was not associated to HIV-1 antigens sensitization. Finally, ART-exposed neonates showed to be born with lower weight, and it was inversely correlated with maternal peripheral TNF-a level. In summary, the data presented here suggest a significant disturbance in cytokine network of HIV-1-uninfected neonates exposed to potent anti-retroviral schemes during pregnancy.