Multitarget Hybrid Fasudil Derivatives as a New Approach to the Potential Treatment of Amyotrophic Lateral Sclerosis.

Hybrid compounds containing structural fragments of the Rho kinase inhibitor fasudil and the NRF2 inducers caffeic and ferulic acids were designed with the aid of docking and molecular mechanics studies. Following the synthesis of the compounds using a peptide-coupling methodology, they were characterized for their ROCK2 inhibition, radical scavenging, effects on cell viability (MTT assay), and NRF2 induction (luciferase assay). One of the compounds (1d) was selected in view of its good multitarget profile and good tolerability. It was able to induce the NRF2 signature, promoting the expression of the antioxidant response enzymes HO-1 and NQO1, via a KEAP1-dependent mechanism. Analysis of mRNA and protein levels of the NRF2 pathway showed that 1d induced the NRF2 signature in control and SOD1-ALS lymphoblasts but not in sALS, where it was already increased in the basal state. These results show the therapeutic potential of this compound, especially for ALS patients with a SOD1 mutation.

Identifying Chemical Composition, Safety and Bioactivity of Thai Rice Grass Extract Drink in Cells and Animals.

Rice grass has been reported to contain bioactive compounds that possess antioxidant and free-radical scavenging activities. We aimed to assess rice grass extract (RGE) drink by determining catechin content, free-radical scavenging and iron-binding properties, as well as toxicity in cells and animals. Young rice grass (Sukhothai-1 strain) was dried, extracted with hot water and lyophilized in a vacuum chamber. The resulting extract was reconstituted with deionized water (260 mg/40 mL) and served as Sukhothai-1 rice grass extract drink (ST1-RGE). HPLC results revealed at least eight phenolic compounds, for which the major catechins were catechin, epicatechin and epigallocatechin-3-gallate (EGCG) (2.71-3.57, 0.98-1.85 and 25.47-27.55 mg/40 mL serving, respectively). Elements (As, Cu, Pb, Sn and Zn) and aflatoxin (B1, B2, G1 and G2) contents did not exceed the relevant limits when compared with WHO guideline values. Importantly, ST1-RGE drink exerted radical-scavenging, iron-chelating and anti-lipid peroxidation properties in aqueous and biological environments in a concentration-dependent manner. The drink was not toxic to cells and animals. Thus, Sukhothai-1 rice grass product is an edible drink that is rich in catechins, particularly EGCG, and exhibited antioxidant, free radical scavenging and iron-binding/chelating properties. The product represents a functional drink that is capable of alleviating conditions of oxidative stress and iron overload.

The catalytic and ROS-scavenging activities of green synthesized, antiferromagneticα-Fe2O3nanoparticle with a prismatic octahedron morphology from pomegranate rind extract.

Phenolic compounds (like 4-nitrophenol) and dyes (like methyl orange) are common by-products discharged by many industries as wastes; they are toxic and may induce discomfort and irritation in humans when ingested. Most of these compounds can be made less toxic through catalytic degradation. Metal oxide nanoparticles are found to have high catalytic activity and can degrade toxic phenolic compounds and dyes. In the current study, pomegranate rind extract was used for the green synthesis of iron oxide nanoparticles that exhibited an octahedron morphology revealed by scanning electron microscopy analysis. Energy dispersive x-ray analysis showed 47.96% content of Fe (by weight); high resolution-transmission electron microscopy analysis confirmed that the nanoparticles had a particle size of 22.54 ± 4.13 nm. The particles were further characterized by x-ray diffraction, fourier transform-infrared spectroscopy, vibrating sample magnetometer, and thermogravimetric analysis. The nanoparticle proved to be efficient in reducing 4-nitrophenol and methyl orange. It was also found to be non-toxic towards murine macrophages, RAW 264.7 with good ROS-scavenging potential compared to control.

Probiotic Administration Mitigates Bisphenol A Reproductive Toxicity in Zebrafish.

Although the use of bisphenol A (BPA) has been banned in a number of countries, its presence in the environment still creates health issues both for humans and wildlife. So far, BPA toxicity has been largely investigated on different biological processes, from reproduction to development, immune system, and metabolism. In zebrafish, Danio rerio, previous studies revealed the ability of environmentally relevant concentrations of this contaminant to significantly impair fertility via epigenetic modification. In addition, several studies demonstrated the ability of different probiotic strains to improve organism health. This study provides information on the role of the probiotic mixture SLAb51 to counteract adverse BPA effects on reproduction. A 28-day trial was set up with different experimental groups: BPA, exposed to 10 µg/L BPA; P, receiving a dietary supplementation of SLAb51 at a final concentration of 109 CFU/g; BPA+P exposed to 10 µg/L BPA and receiving SLAb51 at a final concentration of 109 CFU/g and a C group. Since oocyte growth and maturation represent key aspects for fertility in females, studies were performed on isolated class III (vitellogenic) and IV (in maturation) follicles and liver, with emphasis on the modulation of the different vitellogenin isoforms. In males, key signals regulating spermatogenesis were investigated. Results demonstrated that in fish exposed to the combination of BPA and probiotic, most of the transcripts were closer to C or P levels, supporting the hypothesis of SLAb51 to antagonize BPA toxicity. This study represents the first evidence related to the use of SLAb51 to improve reproduction and open new fields of investigation regarding its use to reduce endocrine disrupting compound impacts on health.

A Co-Doped Fe3O4 Nanozyme Shows Enhanced Reactive Oxygen and Nitrogen Species Scavenging Activity and Ameliorates the Deleterious Effects of Ischemic Stroke.

Acute ischemic stroke has become the major cause of mortality and disability worldwide. Following ischemic stroke, the reperfusion injury is mainly mediated by the burst of reactive oxygen and nitrogen species (RONS). Therefore, blocking the excessive production or removing RONS holds great promise as a potential therapeutic strategy. Herein, we developed a Co-doped Fe3O4 nanozyme that is capable of scavenging H2O2, O2•-, •NO, and ONOO- in vitro and in vivo and provides neuroprotection against ischemic stroke. In vitro experiments showed that pre-incubation with the Co-Fe3O4 nanozyme could prevent neurotoxicity and neuroinflammation induced by H2O2 or lipopolysaccharide, respectively, in HT22 cells. After intravenous administration, the Co-Fe3O4 nanozyme showed no signs of toxicity in peripheral organs of C57BL/6J mice, even after prolonged delivery for 4 weeks. In permanent photothrombotic stroke model and transient middle cerebral artery occlusion stroke model, the Co-Fe3O4 nanozyme specifically accumulated in the infarct rim at 72 h post-stroke and was endocytosed by neurons, astrocytes, microglia, and endothelial cells. Importantly, the Co-Fe3O4 nanozyme delivery reduced the infarct volume in both stroke models. The observation that the Co-Fe3O4 nanozyme was efficacious in two well-characterized ischemic stroke models provides strong evidence that it represents a powerful tool for targeting oxidative and nitrosative stress in the ischemic brain.

Chitosan - Rosmarinic acid conjugates with antioxidant, anti-inflammatory and photoprotective properties.

Rosmarinic acid is an attractive candidate for skin applications because of its antioxidant, anti-inflammatory, and photoprotective functions, however, its poor bioavailability hampers its therapeutic outcome. In this context, synthesis of polymer conjugates is an alternative to enlarge its applications. This work describes the synthesis of novel water-soluble chitosan - rosmarinic acid conjugates (CSRA) that have great potential for skin applications. Chitosan was functionalized with different contents of rosmarinic acid as confirmed by ATR-FTIR, 1H NMR and UV spectroscopies. CSRA conjugates presented three-fold radical scavenger capacity compared to the free phenolic compound. Films were prepared by solvent-casting procedure and the biological activity of the lixiviates was studied in vitro. Results revealed that lixiviates reduced activation of inflamed macrophages, improved antibacterial capacity against E. coli with respect to native chitosan and free rosmarinic acid, and also attenuated UVB-induced cellular damage and reactive oxygen species production in fibroblasts and keratinocytes.

Prussian Blue Nanozymes Prevent Anthracycline-Induced Liver Injury by Attenuating Oxidative Stress and Regulating Inflammation.

Anthracycline-induced liver injury (AILI) is becoming an increasingly serious and potential clinical complication and is linked to reactive oxygen species (ROS) production and subsequent inflammatory response. Herein, we demonstrated that artificial Prussian blue nanozymes (PBZs) prevented daunorubicin-induced liver injury, a prototype of AILI, by attenuating ROS production and regulating inflammation. PBZs exhibited multienzyme activity and could scavenge ROS and free radicals. At the cellular level, PBZs could effectively eliminate ROS, suppress hepatocyte apoptosis, reduce deoxyribonucleic acid damage, and decrease the levels of inflammatory cytokines and chemokines. According to the results of the in vivo study, pretreatment with PBZs also resulted in a desirable protective effect against AILI, as indicated by both a decrease in biochemical indicator levels and hepatocyte necrosis. PBZs upregulated antioxidative genes by activating the Nrf2 pathway to reduce oxidative stress. Meanwhile, PBZs counteracted the inflammatory response based on the decreased expression levels of myeloperoxidase and F4/80 in the liver. Collectively, our findings indicate that PBZ-based nanotherapy is a novel strategy for protecting against AILI.

Maculosin, a non-toxic antioxidant compound isolated from Streptomyces sp. KTM18.

CONTEXT: Streptomyces species are prolific sources of bioactive secondary metabolites known especially for their antimicrobial and anticancer activities. OBJECTIVE: This study sought to isolate and characterize antioxidant molecules biosynthesized by Streptomyces sp. KTM18. The antioxidant potential of an isolated compound and its toxicity were accessed. MATERIALS AND METHODS: The compound was purified using bioassay-guided chromatography techniques. Nuclear magnetic resonance (NMR) experiments were carried out for structure elucidation. The antioxidant potential of the isolated compound was determined using DPPH free radical scavenging assay. The toxicity of the isolated compound was measured using a brine shrimp lethality (BSL) assay. RESULTS: Ethyl acetate extract of Streptomyces sp. KTM18 showed more than 90% inhibition of DPPH free radical at 50 µg/mL of the test concentration. These data were the strongest among 13 Streptomyces isolates (KTM12-KTM24). The active molecule was isolated and characterized as maculosin (molecular formula, C14H16N2O3 as determined by the [M + H]+ peak at 261.1259). The DPPH free radical scavenging activity of pure maculosin was higher (IC50, 2.16 ± 0.05 µg/mL) than that of commercial butylated hydroxyanisole (BHA) (IC50, 4.8 ± 0.05 µg/mL). No toxicity was observed for maculosin (LD50, <128 µg/mL) in brine shrimp lethality assay (BSLA) up to the compound's antioxidant activity (IC50) concentration range. The commercial standard, berberine chloride, showed toxicity in BSLA with an LD50 value of 8.63 ± 0.15 µg/mL. CONCLUSIONS: Maculosin may be a leading drug candidate in various cosmetic and therapeutic applications owing to its strong antioxidant and non-toxic properties.

Investigation into the biological properties, secondary metabolites composition, and toxicity of aerial and root parts of Capparis spinosa L.: An important medicinal food plant.

Capparis spinose L. also known as Caper is of great significance as a traditional medicinal food plant. The present work was targeted on the determination of chemical composition, pharmacological properties, and in-vitro toxicity of methanol and dichloromethane (DCM) extracts of different parts of C. spinosa. Chemical composition was established by determining total bioactive contents and via UHPLC-MS secondary metabolites profiling. For determination of biological activities, antioxidant capacity was determined through DPPH, ABTS, CUPRAC, FRAP, phosphomolybdenum, and metal chelating assays while enzyme inhibition against cholinesterase, tyrosinase, α-amylase and α-glucosidase were also tested. All the extracts were also tested for toxicity against two breast cell lines. The methanolic extracts were found to contain highest total phenolic and flavonoids which is correlated with their significant radical scavenging, cholinesterase, tyrosinase and glucosidase inhibition potential. Whereas DCM extracts showed significant activity for reducing power, phosphomolybdenum, metal chelation, tyrosinase, and α-amylase inhibition activities. The secondary metabolites profiling of both methanolic extracts exposed the presence of 21 different secondary metabolites belonging to glucosinolate, alkaloid, flavonoid, phenol, triterpene, and alkaloid derivatives. The present results tend to validate folklore uses of C. spinose and indicate this plant to be used as a potent source of designing novel bioactive compounds.

Natural Phyto-Antioxidant Albumin Nanoagents to Treat Advanced Alzheimer's Disease.

Phytotherapeutic approaches are of immense value in the treatment of advanced Alzheimer's disease (AD) because of their diverse biological components and potential multitarget mechanisms. In this study, quercetin, a natural neuroprotective flavonoid, was encapsulated in human serum albumin to obtain HSA@QC nanoparticles (HQ NPs) as a natural phyto-antioxidant albumin nanoagent for the treatment of advanced AD. HQ NPs showed excellent antioxidant effects and protected PC12 cells from H2O2-induced oxidative damage. The intranasal administration of HQ NPs in 11-month-old APP/PS1 mice, which represented advanced AD, effectively prevented the loss of body weight, increased survival rates, and significantly reduced oxidative stress, Aß aggregation, neuronal apoptosis, and synaptic damage in the brain. It also ultimately reversed severely impaired cognitive function. In addition to their favorable anti-AD effects, HQ NPs exhibited excellent biosafety and biocompatibility owing to their natural composition and are expected to become an ideal choice for future drug development and clinical applications.

Transcriptome analysis reveals the hepatoprotective mechanism of soybean meal peptides against alcohol-induced acute liver injury mice.

This study aimed was to explore the hepatoprotective potential of soybean meal peptides (SPs) against alcohol-induced liver injury and investigate the underlying mechanisms through transcriptome analysis. The chemical antioxidant analysis of SPs exhibited potent ABTS radical scavenging capacity (11.94 ± 0.41 mg TE/100 mg peptide), ferric reducing antioxidant power (6.42 ± 0.32 mmol Fe2+/100 mg peptide), and oxygen radical absorption capacity (14.78 ± 0.01 mg TE/100 mg peptide). Moreover, SPs increased cell viability and reduced intracellular reactive oxygen species levels in Caco-2 cells by H2O2-induced, and without cytotoxicity. In the mice model, preintervention with SPs reduced the levels of aspartate transaminase/alanine transaminase, total cholesterol, triglyceride and malondialdehyde by alcohol-induced, meanwhile, increased the levels of total superoxide dismutase, glutathione and catalase by alcohol-induced. Histological analysis showed that SPs alleviated the liver injury by alcohol-induced and no toxic effects on the kidneys. According to transcriptome analysis, 1737 genes were significantly differentially expressed (1076 up-regulated and 661 down-regulated) after SPs pretreatment. The main functions of these genes were related to inflammation, lipid metabolism and oxidation. The findings from the present study suggested that SPs produced positive hepatoprotection and showed potential to be used as a dietary supplement or an ingredient of functional food.

Catalytic and highly regenerable aminic organoselenium antioxidants with cytoprotective effects.

Novel N-methylated ebselenamine antioxidants were prepared from the corresponding diselenides with iodomethane. All ebselenamines showed excellent chain-breaking and glutathione peroxidase (GPx)-like activities. They could also inhibit lipid peroxidation much more efficiently than α-tocopherol. They could also mimic the functions of the GPx-enzymes nearly two times better than ebselen in the coupled reductase assay. Also, they were found to scavenge the ROS produced at low concentration (10 µM) with low toxicity effects and could have therapeutic potential against autoxidation. It is anticipated that these compounds could potentially be used against several diseases caused by autoxidation, and thus provide protection from cell death to mammals.

Genotoxic and Cytotoxic Properties of Zinc Oxide Nanoparticles Phyto-Fabricated from the Obscure Morning Glory Plant Ipomoea obscura (L.) Ker Gawl.

The study was undertaken to investigate the antioxidant, genotoxic, and cytotoxic potentialities of phyto-fabricated zinc oxide nanoparticles (ZnO-NPs) from Ipomoea obscura (L.) Ker Gawl. aqueous leaf extract. The UV-visible spectral analysis of the ZnO-NPs showed an absorption peak at 304 nm with a bandgap energy of 3.54 eV, which are characteristics of zinc nanoparticles. Moreover, the particles were of nano-size (~24.26 nm) with 88.11% purity and were agglomerated as observed through Scanning Electron Microscopy (SEM). The phyto-fabricated ZnO-NPs offered radical scavenging activity (RSA) in a dose-dependent manner with an IC50 of 0.45 mg mL-1. In addition, the genotoxicity studies of ZnO-NPs carried out on onion root tips revealed that the particles were able to significantly inhibit the cell division at the mitotic stage with a mitotic index of 39.49%. Further, the cytotoxic studies on HT-29 cells showed that the phyto-fabricated ZnO-NPs could arrest the cell division as early as in the G0/G1 phase (with 92.14%) with 73.14% cells showing early apoptotic symptoms after 24 h of incubation. The results of the study affirm the ability of phyto-fabricated ZnO-NPs from aqueous leaf extract of I. obscura is beneficial in the cytotoxic application.

Supramolecular self-assemblies of engineered polyethylenimines as multifunctional nanostructures for DNA transportation with excellent antimicrobial activity.

In this study, indole-3-butanoic acid (IBA), a biologically and environmentally safe entity, has been grafted onto low and high molecular weight (1.8 and 25 kDa) polyethylenimines (PEI) mainly through primary amines to obtain amphiphilic indole-3-butanoyl-polyethylenimines (IBPs). Two series of IBPs (IBP1.8 and IBP25) were prepared which, on self-assembly in aqueous medium, yielded multifunctional nanomicellar structures (IBP1.8 and IBP25) capable of transporting genetic material in vitro and exhibiting other biological activities. Physicochemical characterization showed the size of IBP1.8 and IBP25 nanostructures in the range of ~332-234 nm and ~283-166 nm, respectively, with zeta potential varying from ~+29-17 mV and ~+37-25 mV. DNA release assay demonstrated higher release of plasmid DNA from IBP nanostructures as compared to native PEIs. Cytotoxicity showed a decreasing pattern with increasing degree of grafting of IBA onto PEIs making these nanostructures non-toxic. pDNA complexes of these nanostructures (both IBPs1.8 and IBPs25) displayed considerably higher transfection efficiency, however, IBP1.8/pDNA complexes performed much better (~7-9 folds) as compared to native PEI/pDNA and Lipofectamine/pDNA complexes on mammalian cells. CLSM analysis revealed that these complexes entered nucleus in sufficient amounts suggesting higher uptake and efficient internalization of the complexes. Besides, these supramolecular nanostructures not only exhibited excellent antimicrobial potential (MIC ~49-100 µg/ml) against clinical as well as resistant pathogenic strains but also found to possess antioxidant property. Overall, the projected low molecular weight PEI-based vectors could serve as more effective multifunctional nanomaterials having promising potential for future gene therapy applications with capability to provide protection against other bacterial infections.

Ceria Nanozymes with Preferential Renal Uptake for Acute Kidney Injury Alleviation.

Rhabdomyolysis-induced acute kidney injury (AKI) is closely related to abundant reactive oxygen species (ROS). Owing to the multi-enzymatic activity and broad-spectrum ROS scavenging capacity of ceria nanoparticles (ceria NPs), herein, we report ultrasmall citric acid modified ceria nanozymes (3-4 nm) as antioxidants to alleviate rhabdomyolysis-induced AKI through removing excessive ROS. The as-prepared ceria NPs exhibited multi-enzymatic properties such as peroxidase, catalase, and superoxide dismutase, offering efficient protection of renal cells against H2O2 stimulation in vitro. Moreover, due to their ultrasmall size, ceria NPs could efficiently accumulate in the kidneys, thus protecting renal cells against ROS in vivo. Our results present ultrasmall ceria nanozymes as antioxidants for rhabdomyolysis-induced AKI alleviation, which shows great potential in clinic.

The effects of Piper sarmentosum aqueous extracts on zebrafish (Danio rerio) embryos and caudal fin tissue regeneration.

In Malaysia, Piper sarmentosum or 'kaduk' is commonly used in traditional medicines. However, its biological effects including in vivo embryonic toxicity and tissue regenerative properties are relatively unknown. The purpose of this study was to determine zebrafish (Danio rerio) embryo toxicities and caudal fin tissue regeneration in the presence of P. sarmentosum aqueous extracts. The phytochemical components and antioxidant activity of the extract were studied using GC-MS analysis and DPPH assay, respectively. Embryo toxicity tests involving survival, heartbeat, and morphological analyses were conducted to determine P. sarmentosum extract toxicity (0-60 µg/mL); concentrations of 0-400 µg/mL of the extract were used to study tissue regeneration in the zebrafish caudal fin. The extract contained several phytochemicals with antioxidant activity and exhibited DPPH scavenging activity (IC50 = 50.56 mg/mL). Embryo toxicity assays showed that a concentration of 60 µg/mL showed the highest rates of lethality regardless of exposure time. Slower embryogenesis was observed at 40 µg/mL, with non-viable embryos first detected at 50 µg/mL. Extracts showed significant differences (p < 0.01) for tissue regeneration at all concentrations when compared to non-treated samples. In conclusion, Piper sarmentosum extracts accelerated tissue regeneration, and extract concentrations at 60 µg/mL showed the highest toxicity levels for embryo viability.

Anti-inflammatory and antioxidant potential, in vivo toxicity, and polyphenolic composition of Eugenia selloi B.D.Jacks. (pitangatuba), a Brazilian native fruit.

Brazilian native fruits are a rich source of polyphenolic compounds that can act as anti-inflammatory and antioxidant agents. Here, we determined the polyphenolic composition, anti-inflammatory mechanism of action, antioxidant activity and systemic toxicity in Galleria mellonella larvae of Eugenia selloi B.D.Jacks. (synonym Eugenia neonitida Sobral) extract (Ese) and its polyphenol-rich fraction (F3) obtained through bioassay-guided fractionation. Phenolic compounds present in Ese and F3 were identified by LC-ESI-QTOF-MS. The anti-inflammatory activity of Ese and F3 was tested in vitro and in vivo through NF-κB activation, cytokine release and neutrophil migration assays. The samples were tested for their effects against reactive species (ROO•, O2•-, HOCl and NO•) and for their toxicity in Galleria mellonella larvae model. The presence of hydroxybenzoic acid, ellagitannins and flavonoids was identified. Ese and F3 reduced NF-κB activation, cytokine release and neutrophil migration, with F3 being three-fold more potent. Overall, F3 exhibited strong antioxidant effects against biologically relevant radicals, and neither Ese nor F3 were toxic to G. mellonella larvae. In conclusion, Ese and F3 revealed the presence of phenolic compounds that decreased the inflammatory parameters evaluated and inhibited reactive oxygen/nitrogen species. E. selloi is a novel source of bioactive compounds that may provide benefits for human health.

Resveratrol diminishes bisphenol A-induced oxidative stress through TRPM2 channel in the mouse kidney cortical collecting duct cells.

Bisphenol A (BisPH-A) is a latent danger that threatens our health, which we frequently exposure in our modern life (e.g. the widespread use of drinking water in plastic pet bottles). But the BisPH-A induced transient receptor potential melastatin 2 (TRPM2)-mediated oxidative stress and apoptosis in these cells has not been studied yet. Calcium (Ca2+) plays an important role in a versatile intracellular signal transduction that works over a wide range to regulate oxidative stress processes. TRPM2 is activated by oxidative stress and it has emerged as an important Ca2+ signaling mechanism in a variety of cells, contributing many cellular functions including cell death. Resveratrol (RESV), which belongs to the polyphenol group, acts as an antioxidant, eliminating cellular oxidative stress and increasing the body's resistance to diseases. The current study aimed to elucidate the effect of antioxidant resveratrol on TRPM2-mediated oxidative stress induced by BisPH-A exposure in the mouse kidney cortical collecting duct cells (mpkCCDcl4). The cells were divided into four groups as control, resveratrol (50 µM for 24 h), BisPH-A (100 µM for 24 h) and BisPH-A + RESV. Intracellular free Ca2+ concentrations and TRPM2 channel currents were high in BisPH-A treated cells, but decreased with resveratrol treatment. In addition, BisPH-A induced mitochondrial membrane depolarization, reactive oxygen species (ROS), caspase 3, caspase 9 and apoptosis values were decreased by the resveratrol treatment. In conclusion, resveratrol protected cells from BisPH-A induced oxidative damage. In this study, we showed that TRPM2 channel mediates this protective effect of resveratrol.

Synthesis, in vitro safety and antioxidant activity of new pyrrole hydrazones.

Six new N-pyrrolylhydrazide hydrazones were synthesized under micro synthesis conditions, assuring about 59-93 % yield, low harmful emissions and reagent economy. The structures of the new compounds were elucidated by melting points, TLC characteristics, IR, 1H and 13C NMR spectral data followed by MS data. The purity of the obtained compounds was proven by the corresponding elemental analyses. "Lipinski's rule of five" parameters were applied for preliminary evaluation of the pharmacokinetic properties of the target molecules. The initial in vitro safety screening for cytotoxicity (on HepG2 cells) and hemocompatibility (hemolysis assay) showed good safety of the new compounds, where ethyl 5-(4-bromophenyl)-1-(1-(2-(4-hydroxy-3-methoxybenzylidene)-hydrazineyl)-1-oxo-3-phenylpropan-2-yl)-2-methyl-1H-pyr-role-3-carboxylate (4d) and ethyl 5-(4-bromophenyl)-1-(1-(2-(2-hydroxybenzylidene)hydrazineyl)-1-oxo-3-phenylpropan--2-yl)-2-methyl-1H-pyrrole-3-carboxylate (4a) were the least toxic. The antioxidant activity in terms of radical scavenging activity (DPPH test) and reducing ability (ABTS) was also evaluated. The antioxidant protective potential of the compounds was next determined in different in vitro cellular-based models, revealing compounds 4d and 3 [ethyl 5-(4-bromophenyl)-1-(1-hydrazineyl-1-oxo-3-phenylpropan-2-yl)-2-methyl-1H-pyrrole-3-carboxylate] as the most promising compounds, with 4d having better safety profile.

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.