Butylated hydroxyanisole induces vascular endothelial injury via TFEB-mediated degradation of GPX4 and FTH1.

Butylated hydroxyanisole (BHA) is one of the most commonly used antioxidants and is widely used in food, but whether it causes vascular damage has not been clearly studied. The present study demonstrated for the first time that BHA reduced the viability of human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (BEND3) in a dose- and time-dependent manner. Moreover, BHA inhibited the migration and proliferation of vascular endothelial cells (ECs). Further analysis revealed that in ECs, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed the BHA-induced increase in Fe2+ and malonaldehyde (MDA) levels. Acridine orange staining demonstrated that BHA increased lysosomal permeability. At the protein level, BHA increased the expression of transcription factor EB (TFEB) and decreased the expression of glutathione peroxidase (GPX4), solute carrier family 7 member 11 (SLC7A11, xCT), and ferritin heavy chain 1 (FTH1). Moreover, these effects of BHA could be reversed by knocking down TFEB. In vivo experiments confirmed that BHA caused elevated pulse wave velocity (PWV) and reduced acetylcholine-dependent vascular endothelial diastole. In conclusion, BHA degrades GPX4, xCT, and FTH1 through activation of the TFEB-mediated lysosomal pathway and promotes ferroptosis, ultimately leading to vascular endothelial cell injury.

Antioxidants Amelioration Is Insufficient to Prevent Acrylamide and Alpha-Solanine Synergistic Toxicity in BEAS-2B Cells.

Cells produce free radicals and antioxidants when exposed to toxic compounds during cellular metabolism. However, free radicals are deleterious to lipids, proteins, and nucleic acids. Antioxidants neutralize and eliminate free radicals from cells, preventing cell damage. Therefore, the study aims to determine whether the antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) will ameliorate the maximum dose of acrylamide and alpha (α)-solanine synergistic toxic effects in exposed BEAS-2B cells. These toxic compounds are consumed worldwide by eating potato products. BEAS-2B cells were simultaneously treated with BHA 10 µM and BHT 20 µM and incubated in a 5% CO2 humidified incubator for 24 h, followed by individual or combined treatment with acrylamide (3.5 mM) and α-solanine (44 mM) for 48 h, including the controls. Cell morphology, DNA, RNA, and protein were analyzed. The antioxidants did not prevent acrylamide and α-solanine synergistic effects in exposed BEAS-2B cells. However, cell morphology was altered; polymerase chain reaction (PCR) showed reduced RNA constituents but not DNA. In addition, the toxic compounds synergistically inhibited AKT/PKB expression and its downstream genes. The study showed BHA and BHT are not protective against the synergetic toxic effects of acrylamide and α-solanine in exposed BEAS-2B cells.

Effect of Administration of an Equal Dose of Selected Dietary Chemicals on Nrf2 Nuclear Translocation in the Mouse Liver.

Certain dietary chemicals influenced the expression of chemopreventive genes through the Nrf2-Keap1 pathway. However, the difference in Nrf2 activation potency of these chemicals is not well studied. This study is aimed at determining the difference in the potency of liver Nrf2 nuclear translocation induced by the administration of equal doses of selected dietary chemicals in mice. Male ICR white mice were administered 50 mg/kg of sulforaphane, quercetin, curcumin, butylated hydroxyanisole, and indole-3-carbinol for 14 days. On day 15, the animals were sacrificed, and their livers were isolated. Liver nuclear extracts were prepared, and Nrf2 nuclear translocation was detected through Western blotting. To determine the implication of the Nrf2 nuclear translocation on the expression levels of several Nrf2-regulated genes, liver RNA was extracted for qPCR assay. Equal doses of sulforaphane, quercetin, curcumin, butylated hydroxyanisole, and indole-3-carbinol significantly induced the nuclear translocation of Nrf2 with different intensities and subsequently increased the expression of Nrf2-regulated genes with an almost similar pattern as the Nrf2 nuclear translocation intensities (sulforaphane > butylated hydroxyanisole = indole-3-carbinol > curcumin > quercetin). In conclusion, sulforaphane is the most potent dietary chemical that induces the Nrf2 translocation into the nuclear fraction in the mouse liver.

Antioxidant, Antidiabetic, Anticholinergic, and Antiglaucoma Effects of Magnofluorine.

Magnofluorine, a secondary metabolite commonly found in various plants, has pharmacological potential; however, its antioxidant and enzyme inhibition effects have not been investigated. We investigated the antioxidant potential of Magnofluorine using bioanalytical assays with 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+), N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD•+), and 1,1-diphenyl-2-picrylhydrazyl (DPPH•) scavenging abilities and K3[Fe(CN)6] and Cu2+ reduction abilities. Further, we compared the effects of Magnofluorine and butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), α-Tocopherol, and Trolox as positive antioxidant controls. According to the analysis results, Magnofluorine removed 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals with an IC50 value of 10.58 µg/mL. The IC50 values of BHA, BHT, Trolox, and α-Tocopherol were 10.10 µg/mL, 25.95 µg/mL, 7.059 µg/mL, and 11.31 µg/mL, respectively. Our results indicated that the DPPH· scavenging effect of Magnofluorine was similar to that of BHA, close to that of Trolox, and better than that of BHT and α-tocopherol. The inhibition effect of Magnofluorine was examined against enzymes, such as acetylcholinesterase (AChE), α-glycosidase, butyrylcholinesterase (BChE), and human carbonic anhydrase II (hCA II), which are linked to global disorders, such as diabetes, Alzheimer's disease (AD), and glaucoma. Magnofluorine inhibited these metabolic enzymes with Ki values of 10.251.94, 5.991.79, 25.411.10, and 30.563.36 nM, respectively. Thus, Magnofluorine, which has been proven to be an antioxidant, antidiabetic, and anticholinergic in our study, can treat glaucoma. In addition, molecular docking was performed to understand the interactions between Magnofluorine and target enzymes BChE (D: 6T9P), hCA II (A:3HS4), AChE (B:4EY7), and α-glycosidase (C:5NN8). The results suggest that Magnofluorine may be an important compound in the transition from natural sources to industrial applications, especially new drugs.

Screening of Carbonic Anhydrase, Acetylcholinesterase, Butyrylcholinesterase, and α-Glycosidase Enzyme Inhibition Effects and Antioxidant Activity of Coumestrol.

Coumestrol (3,9-dihydroxy-6-benzofuran [3,2-c] chromenone) as a phytoestrogen and polyphenolic compound is a member of the Coumestans family and is quite common in plants. In this study, antiglaucoma, antidiabetic, anticholinergic, and antioxidant effects of Coumestrol were evaluated and compared with standards. To determine the antioxidant activity of coumestrol, several methods-namely N,N-dimethyl-p-phenylenediamine dihydrochloride radical (DMPD•+)-scavenging activity, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical (ABTS•+)-scavenging activity, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•)-scavenging activity, potassium ferric cyanide reduction ability, and cupric ion (Cu2+)-reducing activity-were performed. Butylated hydroxyanisole (BHA), Trolox, α-Tocopherol, and butylated hydroxytoluene (BHT) were used as the reference antioxidants for comparison. Coumestrol scavenged the DPPH radical with an IC50 value of 25.95 µg/mL (r2: 0.9005) while BHA, BHT, Trolox, and α-Tocopherol demonstrated IC50 values of 10.10, 25.95, 7.059, and 11.31 µg/mL, respectively. When these results evaluated, Coumestrol had similar DPPH•-scavenging effect to BHT and lower better than Trolox, BHA and α-tocopherol. In addition, the inhibition effects of Coumestrol were tested against the metabolic enzymes acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carbonic anhydrase II (CA II), and α-glycosidase, which are associated with some global diseases such as Alzheimer's disease (AD), glaucoma, and diabetes. Coumestrol exhibited Ki values of 10.25 ± 1.94, 5.99 ± 1.79, 25.41 ± 1.10, and 30.56 ± 3.36 nM towards these enzymes, respectively.

Supplementing rabbit diets with butylated hydroxyanisole affects oxidative stress, growth performance, and meat quality.

Butylated hydroxyanisole (BHA) is a synthetic antioxidant analogous of vitamin E. It is used as a preservative to prevent free radical-mediated oxidation in high-fat foods, and this study's objective was to investigate the effects of BHA on oxidative stress and apoptosis in addition to delineating its efficacy as a growth-promoting feed additive. 60 weaned male rabbits (V-line) were randomly divided into four equal groups: BHA0.0 (control), BHA50, BHA100, and BHA150, administered basal diets with 0.0, 50, 100, and 150 mg BHA/kg of feed for 60 days. Animals were examined for growth performance, markers of oxidative stress and apoptosis, and meat characteristics. Compared to the control group, rabbits receiving BHA-supplemented diets exhibited increases in BW and average daily gain (P < 0.01), where BHA50 and BHA100 groups showed increased muscle content of methionine aspartic acid, serine, and glutamine (P < 0.05). These two groups also exhibited elevated catalase and superoxide dismutase activities and diminished malondialdehyde levels in the liver. Butylated hydroxyanisole upregulated fatty acid synthase gene (FASN), especially in BHA100 animals. Bcl-2-associated X/B-celllymphoma-2 (Bax/Bcl-2) ratio significantly increased in animals receiving higher doses of BHA, and the weight of the liver significantly increased following BHA treatment. Supplementing growing rabbits with lower doses of dietary BHA may promote growth performance and meat quality via maintaining the redox balance. Hence, the 50-100 mg/kg may be recommended as a safe and still effective feed additive as well as an oxidative stress attenuator.

Antioxidant and food additive BHA prevents TNF cytotoxicity by acting as a direct RIPK1 inhibitor.

Butylate hydroxyanisole (BHA) is a synthetic phenol that is widely utilized as a preservative by the food and cosmetic industries. The antioxidant properties of BHA are also frequently used by scientists to claim the implication of reactive oxygen species (ROS) in various cellular processes, including cell death. We report on the surprising finding that BHA functions as a direct inhibitor of RIPK1, a major signaling hub downstream of several immune receptors. Our in silico analysis predicts binding of 3-BHA, but not 2-BHA, to RIPK1 in an inactive DLG-out/Glu-out conformation, similar to the binding of the type III inhibitor Nec-1s to RIPK1. This predicted superior inhibitory capacity of 3-BHA over 2-BHA was confirmed in cells and using in vitro kinase assays. We demonstrate that the reported protective effect of BHA against tumor necrosis factor (TNF)-induced necroptotic death does not originate from ROS scavenging but instead from direct RIPK1 enzymatic inhibition, a finding that most probably extends to other reported effects of BHA. Accordingly, we show that BHA not only protects cells against RIPK1-mediated necroptosis but also against RIPK1 kinase-dependent apoptosis. We found that BHA treatment completely inhibits basal and induced RIPK1 enzymatic activity in cells, monitored at the level of TNFR1 complex I under apoptotic conditions or in the cytosol under necroptosis. Finally, we show that oral administration of BHA protects mice from RIPK1 kinase-dependent lethality caused by TNF injection, a model of systemic inflammatory response syndrome. In conclusion, our results demonstrate that BHA can no longer be used as a strict antioxidant and that new functions of RIPK1 may emerge from previously reported effects of BHA.

Antioxidant properties of eugenol, butylated hydroxylanisole, and butylated hydroxyl toluene with key biomolecules relevant to Alzheimer's diseases-In vitro.

This research work examined and likened effect of eugenol a natural phenolic compound with butylated hydroxylanisole (BHA) and butylated hydroxyl toluene (BHT) synthetic phenolic compounds with key biomolecules [acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and monoamine oxidase (MAO)] relevant to Alzheimer's diseases (AD) in vitro. Ten millimolar each sample was prepared in a mixture of ethanol and water (1:1 v/v), and the interactions with AChE, BChE, and MAO were evaluated. Still, ferric reducing antioxidant property, ABTS radicals scavenging ability and lipid peroxidation were carried out. The results revealed eugenol, BHT, and BHA inhibited AChE, BChE, and MAO activities dose-dependently. Though, eugenol had greater inhibitory effect against AChE and BChE activities. Also, eugenol demonstrated higher antioxidant potential compared to BHT and BHA. The potent enzymatic inhibitory and antioxidant effects of eugenol indicate eugenol could be promising as an alternative food additive and neuromodulator in AD management. PRACTICAL APPLICATION: BHT and BHA are synthetic antioxidant employed industrially as food preservative. BHT and BHA are employed in food packaging, drugs, and cosmetics. Although BHT and BHA are widely in use but have been found were associated with alteration in sleeping, induced changes in brain serotonin and norepinephrine levels with increased cholinesterase activity. Endocrine disrupting effects, reproductive disorder is more side effects associated with the use of BHT and BHA. However, eugenol a natural compound found in plants compares favorably with BHT and BHA as antioxidant with many more health promoting benefits such as neuroprotective effects, antiapoptotic effects, and prevent aluminum toxicity. Eugenol being a natural antioxidant with no side effects showing more promising effects over the synthetic phenolic compounds and could be an alternative for the BHT and BHA.

Synthesis of substituted N-(2'-nitrophenyl)pyrrolidine-2-carboxamides towards the design of proline-rich antimicrobial peptide mimics to eliminate bacterial resistance to antibiotics.

The treatment of diseases is under threat due to the increasing resistance of disease-causing bacteria to antibiotics. Likewise, free radical-induced oxidative stress has been implicated in several human disease conditions, such as cancer, stroke and diabetes. In the search for amino acid analogues with antibacterial and antioxidant properties as possible mimics of antimicrobial peptides, substituted N-(2'-nitrophenyl)pyrrolidine-2-carboxamides 4a-4k and N-(2'-nitrophenyl)piperidine-2-carboxamides 4l-4n have been synthesized via a two-step, one-pot amidation of the corresponding acids, using thionyl chloride with different amines in dichloromethane. The carboxamides were characterized by infrared and nuclear magnetic resonance spectroscopy, mass spectrometry and elemental analysis. Carboxamides 4a-4n were assayed against five Gram-positive and five Gram-negative bacterial strains using the broth micro-dilution procedure and compared to standard antibiotic drugs (streptomycin and nalidixic acid). 4b showed the highest antibacterial activity with a minimum inhibitory concentration (MIC) value of 15.6 µg/mL against Staphylococcus aureus. Pertinently, 4b and 4k are promising candidates for narrow-spectrum (Gram-positive) and broad-spectrum antibiotics, respectively. The antioxidant properties of the carboxamides were also evaluated using the 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical cation. 4a and 4k recorded the lowest IC50 values of 1.22 × 10-3 mg/mL (with DPPH) and 1.45 × 10-4 mg/mL (with ABTS), respectively. Notably, 4k recorded about 2.5 times better antioxidant capacity than the positive controls - ascorbic acid and butylated hydroxyanisole. These results bode well for N-aryl carboxamides as good mimics and substitutes for antimicrobial peptides towards mitigating bacterial resistance to antibiotics as well as ameliorating oxidative stress-related diseases.

Comparative study of rosemary extracts and several synthetic and natural food antioxidants. Relevance of carnosic acid/carnosol ratio.

The antiradical power, at equal concentrations of active principles, of the following antioxidants were studied using the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay: butylated-hydroxyanisole, butylated-hydroxytoluene, tert-butylhydroquinone, ascorbyl palmitate, tocopherol, grape seed extract, olive extract and five rosemary extracts with different concentrations of carnosic acid (CA) and carnosol (COL). The reaction kinetics of DPPH scavenging activity in each studied substance identified significant variations in the time needed to reach the steady state. Rosemary extracts were seen to be more effective than the other compounds. CA had higher antioxidant activity than COL, although COL seemed to react faster with DPPH. The relevance of the CA/COL ratio for the antioxidant activity of rosemary extracts was also analysed. The presence of COL in rosemary extracts increased the antioxidant activity with an optimal CA/COL ratio of 2.5-3.0. Olive extract and grape seed extract seem to be very promising additives for use as technological antioxidants.

Effect of natural antioxidants on lipid oxidation in mayonnaise compared with BHA, the industry standard.

INTRODUCTION: Mayonnaise is an oil in water emulsion (O/W) consisting 70-80% oil. Lipid oxidation is a major cause of quality deterioration in mayonnaise. The effectiveness of antioxidants in a hetrophasic systems is highly dependent on their polarity and partitioning properties. OBJECTIVES: The aim of the present study was to determine the effect of a hydrophilic [green tea extract (GTE)] and a lipophilic [tocopherol mixture (TOC)] and BHA on lipid oxidation in mayonnaise during 60 days of storage at 38 °C and to examine the interactions between GTE and TOC, to determine possible synergistic or antagonistic effects in antioxidant activity. METHODS: The oxidative stability was studied by measuring hydroperoxides, volatile organic compounds (VOCs) and colour of mayonnaise during storage. Comprehensive analysis of VOCs was done by static headspace extraction and separation by two-dimensional gas chromatography time of flight mass spectrometry. Sensory analysis was also carried out to study the effect of storage time and antioxidant type on sensory properties of mayonnaise and to investigate the predictive ability of volatile compounds for sensory terms. RESULTS AND CONCLUSION: Addition of GTE (500 ppm) and TOC (500 ppm) increased the formation of hydroperoxides and certain VOCs. The combination of GTE with TOC improved the antioxidant efficacy compared to the individual extracts. However, sensory evaluation demonstrated that GTE promoted the development of unpleasant fishy and rancid aroma. The volatile compound methional, was significantly and positively correlated with fishy and rancid flavour. Regarding colour analysis, GTE showed the highest increase in darkening and browning during storage.

Mkp-1 is required for chemopreventive activity of butylated hydroxyanisole and resveratrol against colitis-associated colon tumorigenesis.

Many dietary compounds show promising protective activity against colon cancer by activating nuclear factor-erythroid 2 related factor 2 (Nrf2). Recently, we reported that mitogen-activated protein kinase phosphatase 1 (Mkp-1) exhibits crosstalk with the Nrf2 signaling pathway, protecting against intestinal inflammation. Here, we present evidence that Mkp-1 is required for the chemopreventive action of the Nrf2 activators butylated hydroxyanisole (BHA) and resveratrol (RSV). In an azoxymethane/dextran sulfate sodium model of colitis-associated tumorigenesis, Mkp-1-/- mice exhibited a phenotype similar to Nrf2-/- mice with significantly more tumors than WT mice. Tumors from Mkp-1-/- mice exhibited higher levels of macrophage infiltration than those from WT mice. This was accompanied by increased expression of nitrotyrosine and p53BP1, markers of oxidative stress and DNA damage, respectively. Moreover, dietary suppression of tumorigenesis using BHA (0.5%) or RSV (300 ppm) supplementation was achieved in WT but not in Mkp-1-/- mice. In adenomas from WT mice, the expression of Mkp-1 was markedly lower than in adjacent normal tissue, concomitant with the down-regulation of Nrf2 and its target genes. Our data revealed that Mkp-1 is required in the protective role of Nrf2 signaling against colitis-associated tumorigenesis.

Comparative Genomic Screen in Two Yeasts Reveals Conserved Pathways in the Response Network to Phenol Stress.

Living organisms encounter various perturbations, and response mechanisms to such perturbations are vital for species survival. Defective stress responses are implicated in many human diseases including cancer and neurodegenerative disorders. Phenol derivatives, naturally occurring and synthetic, display beneficial as well as detrimental effects. The phenol derivatives in this study, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and bisphenol A (BPA), are widely used as food preservatives and industrial chemicals. Conflicting results have been reported regarding their biological activity and correlation with disease development; understanding the molecular basis of phenol action is a key step for addressing issues relevant to human health. This work presents the first comparative genomic analysis of the genetic networks for phenol stress response in an evolutionary context of two divergent yeasts, Schizosaccharomyces pombe and Saccharomyces cerevisiae Genomic screening of deletion strain libraries of the two yeasts identified genes required for cellular response to phenol stress, which are enriched in human orthologs. Functional analysis of these genes uncovered the major signaling pathways involved. The results provide a global view of the biological events constituting the defense process, including cell cycle arrest, DNA repair, phenol detoxification by V-ATPases, reactive oxygen species alleviation, and endoplasmic reticulum stress relief through ergosterol and the unfolded protein response, revealing novel roles for these cellular pathways.

Microencapsulated food-grade antioxidant applied as a preservative of peanut seed quality in microcosm- and pilot-scale trials.

BACKGROUND: In Argentina, peanuts are stored for 3-6 months. It is important to avoid proliferation of fungi and insect pests during this period. In this study, the potential of butylated hydroxyanisole (BHA) microcapsules to conserve peanut kernels was evaluated in microcosms and on a pilot scale. RESULTS: In microcosm assays, microcapsules containing BHA at a dose of 1802 µg g-1 reduced 37% of total fungal count. Higher reductions (77-100%) were obtained with a combined treatment with BHA formulation (1802 µg g-1 ) plus fungicide (methyl thiophanate 0.0100 g L-1  and metalaxyl 0.0133 g L-1 ). However, germination levels of peanut seeds treated with the BHA formulation were less than 6% throughout the incubation time. In pilot-scale trials, the storage conditions allowed the control of fungal development and insect proliferation. Quantifiable levels of BHA were also detected throughout the entire storage period. The combined treatment significantly reduced fungal contamination at 2 months of storage (C1-2015: 37.41%; C1-2016: 28.48%; C2-2016: 45.02%). Seed germination of unshelled stored peanuts was not affected by the formulation. CONCLUSION: The application of the BHA formulation during storage combined with pre-seeding treatment could be an appropriate strategy to maintain the quality of the peanut kernels destined for seed. © 2018 Society of Chemical Industry.

Effect of isoespintanol isolated from Oxandra cf. xylopioides against DNA damage of human lymphocytes.

Synthetic antioxidants are used in the food and pharmaceutical industry, however, there is concern about their safety; this has prompted the search for new antioxidants that are effective, safe and act at low concentrations. The objective of this study is to evaluate the oxygen radical scavenging capacity and clastogenic effect of the Isoespintanol /2-isopropyl-3,6-dimethyl-5-methylphenol) in DNA of human lymphocyte compared with the BHA (Butylated hydroxyanisole). The oxygen radical scavenging ability was evaluated by methods ORACFL and ORACPGR, genotoxicity was determined by comet assay and data analysis was performed using ANOVA and Duncan test. The results show that the oxygen radical scavenging capacity of the BHA is higher than Isoespintanol, however according to the reactivity concept proposed by Lopez-Alarcon and Lissi, the Isoespintanol it is more reactive than BHA. Furthermore, according to some studies, BHA presented adverse effects on the health of consumers. Comet assay results revealed that at concentrations between 3 and 1620 µM the Isoespintanol don't show clastogenic effects on DNA. In conclusion, the antioxidant capacity for the BHA is higher than Isoespintanol, but considering reactivity concepts proposed by López-Alarcon and Lissi, the Isoespintanol is faster to neutralize radicals that the BHA, furthermore, according to the National Institute of Health "BHA" is a human carcinogen.

Negative chronotropism, positive inotropism and lusitropism of 3,5-di-t-butyl-4-hydroxyanisole (DTBHA) on rat heart preparations occur through reduction of RyR2 Ca2+ leak.

3,5-Di-t-butyl-4-hydroxyanisole (DTBHA) is considered as an activator of the skeletal muscle sarcoplasmic reticulum (SR) Ca2+-uptake, endowed with antioxidant and L-type Ca2+ channel blocking activities. In this study we assessed the cardiac effects of DTBHA on Langendorff perfused rat hearts, isolated rat atria and rat cardiac SR membrane vesicles, as well as on several SERCA isoforms of membrane preparations. Moreover, in order to clarify its molecular mechanism of action Ca2+ imaging experiments were carried out on HEK293 cells transiently transfected with RyR2 channel. Docking of DTBHA at the rat RyR2 protein was investigated in silico. In Langendorff perfused rat hearts, DTBHA significantly increased, in a concentration-dependent manner, left ventricular pressure and diastole duration, while reducing heart rate and the time-constant of isovolumic relaxation, leaving unaltered coronary perfusion pressure. At the maximum concentration tested (30 µM), it significantly prolonged PQ interval, but left the corrected QT intervals unaffected. In spontaneously beating atria, DTBHA decreased sinus rate in a concentration-dependent manner. DTBHA, at concentrations higher than 10 µM, increased Ca2+ uptake in cardiac SR without affecting Ca2+-dependent ATPase activity assayed on several SERCA isoforms. Moreover, DTBHA antagonized thapsigargin-stimulated Ca2+ leak in cardiac SR and reduced caffeine-induced, RyR2-activated Ca2+ release in RyR2 expressing HEK293 cells. Using computational approaches, DTBHA showed a good affinity outline into binding sites of RyR2 protein. In conclusion, DTBHA behaved like a negative chronotropic, a positive inotropic and a lusitropic agent on rat heart preparations and improved cardiac SR Ca2+ uptake by lowering SR Ca2+ leak.

Biologically and chemically important hydrazino-containing imidazolines as antioxidant agents.

Biologically and chemically useful hydrazinoimidazolines were evaluated as antioxidant and antihaemolytic agents. 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH•), galvinoxyl radical (GOR), nitric oxide (NO) and hydrogen peroxide (H2O2) scavenging assays, ferric ions reducing power assay, and ex vivo model of rat erythrocytes exposed to 2,2'-azobis(2-methylpropionamidine)dihydrochloride (AAPH) or H2O2 were used. The most potent DPPH• scavengers proved to be hydrazinoimidazolines 3, 2, and 4, revealing excellent antiradical effects - superior or comparable to that of all antioxidant standards used. Moreover, these molecules showed strong NO neutralising potencies - better to that of ascorbic acid (AA) (3), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (3 and 2), butylated hydroxytoluene (BHT) (3 and 2), and butylated hydroxyanisole (BHA) (3, 2, and 4). Compound 4 was also effective in GOR scavenging. The excellent scavenger of GOR, NO, and H2O2 proved to be structure 5, with the potency superior or comparable to the majority of antioxidant standards used. In turn, compound 9 was effective in H2O2 and GOR neutralisation. All hydrazinoimidazolines revealed the reducing power that is higher than BHT. Moreover, the protective effects of most test compounds on oxidatively stressed erythrocytes were observed. Some structure-activity relationships were disclosed. A significance of the primary hydrazino group on antioxidant effects was confirmed. The most likely DPPH• and GOR scavenging mechanisms for test compounds were propound. Among all the investigated molecules, hydrazinoimidazolines 5, 3, 2, 4, and 9, due to their excellent or good antiradical activities, can represent promising antioxidant candidates with prospective utility for prevention of diseases related to reactive oxygen/nitrogen species.

Respiratory syncytial virus infection up-regulates TLR7 expression by inducing oxidative stress via the Nrf2/ARE pathway in A549 cells.

In order to better understand the early pathways of the pathogenesis of, and immune response to, RSV, herein, we explored the relationship between TLR7 expression and oxidative stress induction following RSV infection in A549 cells. We studied the intervening effects of the Nrf2/ARE pathway agonist butylated hydroxyanisole (BHA) and inhibitor trigonelline (TRI) on TLR7 modulation or oxidative stress induction. For comparison purposes, we set up seven treatment groups in this study, including RSV-treated cells, BHA + RSV-treated cells, TRI + RSV-treated cells, normal cell controls, inactivated RSV controls, BHA controls and TRI controls. We measured changes in TLR7, IL-6, TNF-α mRNA using RT-PCR and IL-6, TNF-α and IL-1ß protein using ELISA as well as TLR7, Nrf2 and HO-1 protein using Western blot in A549 cells from the different treatment groups. We also assessed changes in cell proliferation and measured changes in ·OH and NO in A549 cells from the different treatment groups. The results indicate that TLR7 up-regulation is related to RSV infection and the induction of oxidative stress and that TLR7 expression was mediated by the anti-inflammatory effects of Nrf2/ARE pathway inhibitors or agonists. Our experiments may help elucidate the underlying pathology of RSV infection and suggest potential therapeutic targets for drug development and the prevention of RSV-induced human diseases.

The Study of Anti-/Pro-Oxidant, Lipophilic, Microbial and Spectroscopic Properties of New Alkali Metal Salts of 5-O-Caffeoylquinic Acid.

Lithium, sodium, potassium, rubidium and caesium salts of 5-O-caffeoylquinic acid (chlorogenic acid, 5-CQA) were synthesized and described by FT-IR (infrared spectroscopy), FT-Raman (Raman spectroscopy), UV (UV absorption spectroscopy), ¹H (400.15 MHz), 13C (100.63 MHz) NMR (nuclear magnetic resonance spectroscopy). The quantum-chemical calculations at the B3LYP/6-311++G** level were done in order to obtain the optimal structures, IR spectra, NBO (natural bond orbital) atomic charges, HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) orbitals and chemical reactivity parameters for 5-CQA and Li, Na and K 5-CQAs (chlorogenates). The DPPH (α, α-diphenyl-ß-picrylhydrazyl) and FRAP (ferric reducing antioxidant power) assays were used for the preliminary estimation of the antioxidant properties of alkali metal chlorogenates and chlorogenic acid. In the DPPH assay the EC50 parameter were equal to 7.39 µM for 5-CQA and was in the range of 4.50-5.89 µM for salts. The FRAP values for two different concentrations (5 and 2.5 µM) of the studied compounds were respectively 114.22 and 72.53 µM Fe2+ for 5-CQA, whereas for salts they were 106.92-141.13 and 78.93-132.00 µM Fe2+. The 5-CQA and its alkali metal salts possess higher antioxidant properties than commonly applied antioxidants (BHA, BHT, l-ascorbic acid). The pro-oxidant action of these compounds on trolox oxidation was studied in the range of their concentration 0.05-0.35 µM. The lipophilicity (logkw) of chlorogenates and chlorogenic acid was determined by RP-HPLC (reverse phase-high performance liquid chromatography) using five different columns (C8, PHE (phenyl), CN (cyano), C18, IAM (immobilized artificial membrane)). The compounds were screened for their in vitro antibacterial activity against E. coli, Bacillus sp., Staphylococcus sp., Streptococcus pyogenes and antifungal activity against Candida sp. The 5-CQA possessed lower antibacterial (minimal inhibitory concentration, MIC = 7.06 mM) and antifungal (MIC = 14.11 mM) properties than its alkali metal salts (MIC values: 6.46-2.63 mM and 12.91-5.27mM, respectively). The synthesized chlorogenates possessed better antioxidant, lipophilic, antimicrobial as well as lower pro-oxidant properties than the ligand alone. Moreover, a systematic change of the activity of alkali metal salts along the series Li→Cs suggests that there are correlations between the studied biological properties. The type of metal cation in the carboxylate group of chlorogenate is crucial for the activity of studied compounds.

Comparative study of the influence of hawthorn (Crataegus monogyna) berry ethanolic extract and butylated hydroxylanisole (BHA) on lipid peroxidation, myoglobin oxidation, consistency and firmness of minced pork during refrigeration.

BACKGROUND: Following public concern on the use of synthetic food antioxidants, there is an increasing demand for the application of mixed or purified natural antioxidants to maintain quality of meat products quality during storage. The aim of this research was to investigate the effect of ethanolic extract of hawthorn berry, compared to butylated hydroxylanisole (BHA), on lipid peroxidation, myoglobin oxidation, protein electrophoresis pattern, consistency and firmness of minced pork during refrigeration at 4 °C, and to identify the relationship between chemical modifications and consistency variation. RESULTS: After 6 days of refrigeration it was found that the thiobarbituric acid reactive substances value of minced pork containing 200 mg GAE kg-1 total phenolics in minced meat (200 HP) was significantly lower (0.1543 ± 0.006 mg) compared to BHA-treated meat. The ratio of oxymyoglobin to metmyoglobin in treated minced pork was respectively 0.845 for 200 HP and 0.473 for BHA-treated minced meat. Concentrations of 100 HP or 300 HP will generate statistically higher firmness than BHA in minced pork. CONCLUSION: Hawthorn berry ethanolic extract was more effective than BHA in reducing lipid oxidation and protein degradation, for maintaining firmness and consistency of minced pork during 6 days of refrigeration at 4 °C. © 2017 Society of Chemical Industry.