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A series of indole-1,4-disubstituted-1,2,3-triazole conjugates were synthesised by click chemistry. The haemolytic properties and cytoprotective activity of all the newly synthesised indole-triazole conjugates were tested in vitro. In addition, molecular docking was performed in silico for the selected conjugates to determine their antibacterial and antifungal properties. The results indicate that indole-triazole derivatives effectively protect human erythrocytes against free radical-induced haemolysis in a structure-dependent manner and that bis-indole-bis-triazole derivatives with alkyl linkers are excellent cytoprotective agents against oxidative haemolysis. The tested series of indole-1,4-disubstituted-1,2,3-triazole conjugates may have an affinity for the active sites of specific protein domains (PDB IDs: 2Q85 and 5V5Z) according to molecular docking studies. [ABSTRACT FROM AUTHOR]

Chitosan-based films show great potential in terms of application in food preservation and are also promising carriers of biologically active ingredients. This paper presents the potential use of chitosan-based films with the addition of essential oil components, e.g., carvacrol, eugenol, and isoeugenol, intended for food packaging. The characteristics of the obtained films were determined, including antibacterial, mechanical, barrier, and structural parameters. In addition, the antibacterial and antioxidant effects of the essential oil components were assessed. Eugenol (44.41%) and isoeugenol (43.56%) showed high antiradical activities, similar to the activity of Trolox (44.33%), which is used as a standard antioxidant. In turn, carvacrol was characterized by the strongest effect against the examined strains of bacteria, both Gram-positive and Gram-negative. The chitosan film with carvacrol showed the most valuable antibacterial and mechanical properties (tensile strength and elongation at break). The antibacterial activities of the chitosan–carvacrol films were higher than that of the carvacrol solution. The inhibition zones of the chitosan–carvacrol films were in the range 29–41 mm (except for Enterococcus faecalis, with an inhibition zone of 15 mm) compared to the inhibition zones of the carvacrol solution (28 mm). The results showed that chitosan is an effective carrier of fragrance compounds, mainly carvacrol. However, all the tested chitosan-based films with the addition of fragrance compounds showed appropriate parameters (biological, mechanical, and barrier), which makes them an ecological alternative to plastics intended for food packaging. [ABSTRACT FROM AUTHOR]

The ability of the indole–imidazole hybrid ligands to coordinate with the Zn(II) ion and the resulting structures of this new class of coordination compounds were analyzed in order to determine their structural properties and biological functionalities. For this purpose, six novel Zn(II) complexes, [Zn(InIm)2Cl2] (1), [Zn(InMeIm)2Cl2] (2), [Zn(IniPrIm)2Cl2] (3), [Zn(InEtMeIm)2Cl2] (4), [Zn(InPhIm)2Cl2] (5) and [Zn2(InBzIm)2Cl2] (6) (where InIm is 3-((1H-imidazol-1-yl)methyl)-1H-indole), were synthesized by the reactions of ZnCl2 and the corresponding ligand in a 1:2 molar ratio in methanol solvent at an ambient temperature. The structural and spectral characterization of these complexes was performed using NMR, FT–IR and ESI–MS spectrometry and elemental analysis, and the crystal structures of 1–5 were determined using single-crystal X-ray diffraction. Complexes 1–5 form polar supramolecular aggregates by utilizing, for this purpose, the N-H(indole)∙∙∙Cl(chloride) intermolecular hydrogen bonds. The assemblies thus formed differ depending on the distinctive molecular shape, which can be either compact or extended. All complexes were screened for their hemolytic, cytoprotective, antifungal, and antibacterial activities. The results show that the cytoprotective activity of the indole/imidazole ligand significantly increases upon its complexation with ZnCl2 up to a value comparable with the standard antioxidant Trolox, while the response of its substituted analogues is diverse and less pronounced. [ABSTRACT FROM AUTHOR]

Intracellular calcium is maintained at very low concentrations through the action of PMCA Ca++ extrusion pumps. Although much of our knowledge about these Ca++ extrusion pumps derives from studies with human erythrocytes, kinetic studies of Ca++ transport for these cells are limited to radioisotope flux measurements. Here, we developed a robust, microplate-based assay for erythrocyte Ca++ efflux using extracellular fluorescent Ca++ indicators. We optimized Ca++ loading with the A23187 ionophore, established conditions for removal of the ionophore, and adjusted fluorescent dye sensitivity by addition of extracellular EGTA to allow continuous tracking of Ca++ efflux. Efflux kinetics were accelerated by glucose and inhibited in a dose-dependent manner by the nonspecific inhibitor vanadate, revealing that Ca++ pump activity can be tracked in a 384-well microplate format. These studies enable radioisotope-free kinetic measurements of the Ca++ pump and should facilitate screens for specific inhibitors of this essential transport activity. [ABSTRACT FROM AUTHOR]

The transmembrane protein CD47, an innate immune checkpoint protein, plays a pivotal role in preventing healthy erythrocytes from immune clearance. Our study utilized stochastic optical-reconstruction microscopy (STORM) and single-molecule analysis to investigate the distribution of CD47 on the human erythrocyte membrane. Contrary to previous findings in mouse erythrocytes, we discovered that CD47 exists in randomly distributed monomers rather than in clusters across the human erythrocyte membrane. Using 2nd antibody-induced crosslinking, we found that CD47 aggregates into stable clusters within minutes. By comparing these STORM results with those of the fully mobile protein CD59 and the cytoskeleton-bound membrane protein glycophorin C under similar conditions, as well as devising two-color STORM co-labeling and co-clustering experiments, we further quantitatively revealed an intermediate, self-limiting clustering behavior of CD47, elucidating its fractional (∼14%) attachment to the cytoskeleton. Moreover, we report reductions in both the amount of CD47 and its clustering capability in aged erythrocytes, providing new insight into erythrocyte senescence. Together, the combination of STORM and 2nd antibody-based crosslinking unveils the unique self-limiting clustering behavior of CD47 due to its fractional cytoskeleton attachment., (© The Author(s) 2024. Published by Oxford University Press on behalf of Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.)

Introduction: The oxidative stress can affect the biological membranes of different cellular types in the organism, which has been evidenced in the damages to the tissues and organs of patients with COVID-19, reason why the recent investigations are related to the search of cytoprotector and antioxidant drugs that minimize these damages. Objective: To evaluate the human erythrocytes as pharmacological biomodel of antioxidant cytoprotection. Methods: The cytotoxicity pattern was evaluated in erythrocytes induced by peroxide of hydrogen and the system of diagnosis proposed was valued in a cytoprotection assay in erythrocytes, with the use of ascorbic acid as reference substance. Results: For the concentration of erythrocytes used a cytotoxicity model was achieved to the concentration of 10 mM of peroxide at 30 minutes of incubation. The substance of reference used didn't show cytotoxicity signs in the hemolysis test. In the cytoprotection assay a pharmacological effect of the referent was evidenced, with a value of the cytoprotection index of 12.71 µg/mL. The study of optic microscopy showed severe morphological damages in the erythrocytes treated with peroxide of spherocytes, echinocytes and spheroechinocytes type that significantly diminished in presence of this reference substance. Conclusions: The proposed pharmacological biomodel can be used in the evaluation of new therapeutic alternatives with antioxidant cytoprotector properties for the treatment of patients with COVID-19. [ABSTRACT FROM AUTHOR]

• C. citratus, S. spontaneum, O. compressa and D. annulatum showed highest antihemolytic potential. • In vitro ferric thiocyanate and thiobarbituric acid tests were applied. • Among essential elements, ICP-MS revealed highest concentration of sodium in C. ciliaris and potassium in P. annua. • Elements have substantial influence on the biological activities of examined species. Free radicals produced in vivo increases 'oxidative stress' and leads to lipid peroxidation, DNA damage and other disorders such as hemolysis. Present study aimed at evaluating the biological potentials of seventeen Poaceae species in order to scientifically confirm their traditional uses and to identify elements involved in regulating reactive oxygen species. Extracts of seventeen grass species were prepared to determine antioxidant activity using ferric thiocyanate and thiobarbituric acid assays. Sorghum halepense, Eleusine indica, Ochthocloa compressa, Saccharum spontaneum, Dactyloctenium aegyptium and Cymbopogon citratus showed highest antioxidant potential. Antihemolytic potential was assessed by hydrogen peroxide (H 2 O 2) induced hemolysis in human erythrocytes and was found to be highest in C. citratus (96%) followed by S. spontaneum (95%), O. compressa (66%) and D. annulatum (60%). DNA damage activity was performed using p1391Z plasmid DNA. In the end, samples were digested using nitric acid and the concentration of twenty elements was determined using inductively coupled plasma mass spectrometry (ICP-MS) method. Principal component analysis (PCA) of essential elements showed 70% variability and non-essential elements displayed 52% in total variance. Hence, the selected botanicals can be used commercially in the treatment of oxidative-stress related diseases as the concentration of different elements validate their safety and efficacy for human body. [ABSTRACT FROM AUTHOR]

The new series of triazole-bearing gramine derivatives were synthesized through a CuAAC procedure. The structures of all newly obtained compounds were confirmed by spectroscopic analysis and DFT methods. The obtained derivatives were screened for their protective potency against oxidative haemolysis induced by free radicals generated from 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). Our work demonstrates that derivatives with propyl or octyl linker and phthalimide group associated with indole-triazole moiety, which have a folded structure, effectively protect human erythrocytes against oxidative stress-induced haemolysis. [ABSTRACT FROM AUTHOR]

The ability of the indole–imidazole hybrid ligands to coordinate with the Zn(II) ion and the resulting structures of this new class of coordination compounds were analyzed in order to determine their structural properties and biological functionalities. For this purpose, six novel Zn(II) complexes, [Zn(InIm)2Cl2] (1), [Zn(InMeIm)2Cl2] (2), [Zn(IniPrIm)2Cl2] (3), [Zn(InEtMeIm)2Cl2] (4), [Zn(InPhIm)2Cl2] (5) and [Zn2(InBzIm)2Cl2] (6) (where InIm is 3-((1H-imidazol-1-yl)methyl)-1H-indole), were synthesized by the reactions of ZnCl2 and the corresponding ligand in a 1:2 molar ratio in methanol solvent at an ambient temperature. The structural and spectral characterization of these complexes was performed using NMR, FT–IR and ESI–MS spectrometry and elemental analysis, and the crystal structures of 1–5 were determined using single-crystal X-ray diffraction. Complexes 1–5 form polar supramolecular aggregates by utilizing, for this purpose, the N-H(indole)∙∙∙Cl(chloride) intermolecular hydrogen bonds. The assemblies thus formed differ depending on the distinctive molecular shape, which can be either compact or extended. All complexes were screened for their hemolytic, cytoprotective, antifungal, and antibacterial activities. The results show that the cytoprotective activity of the indole/imidazole ligand significantly increases upon its complexation with ZnCl2 up to a value comparable with the standard antioxidant Trolox, while the response of its substituted analogues is diverse and less pronounced.

The aim of the present study was to investigate the effects of honey against aluminium (Al) and hypotonic pressure-induced hemolysis of human red blood cells (RBCs) and hemoglobin degradation in vitro. Human RBCs were pretreated separately with honey samples and ascorbic acid. Four different treatment groups were considered: untreated RBCs (negative control), aluminium treated RBCs (positive control), honey + aluminium treated RBCs, and ascorbic acid + aluminium treated RBCs. Samples were then evaluated by simultaneous measurement of cellular turbidity and hemoglobin (Hb). The results showed that RBCs suspensions treated with honey samples, particularly honey 2 presented highest cell and Hb values compared to that of ascorbic acid and positive control, whereas, ascorbic acid exhibited a prooxidant effect on cell and Hb. The results of this study suggest that honey not only protect cell integrity but also prevent oxidative degradation of Hb. Therefore, the present study demonstrated a protective effect of honey against Al-induced red blood cells hemolysis and hemoglobin degradation. [ABSTRACT FROM AUTHOR]

ABSTRACT Pathogenic intracellular mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium avium, which cause lung diseases, can grow in macrophages. Extracellular mycobacteria have been reported in the lungs, blood, and sputum of patients, indicating the involvement of these pathogens in disease progression. Erythrocytes are involved in the symptoms associated with pulmonary mycobacterial diseases, such as bloody sputum and hemoptysis; however, little attention has been paid to the role of erythrocytes in mycobacterial diseases. Herein, we found that Mycobacterium avium subsp. hominissuis (MAH) and Mycobacterium intracellulare colocalized with erythrocytes at the sites of lung infection, inside capillaries and necrotic areas of granulomas, using histopathological examinations. Electron microscopy showed that MAH adhered and entered human erythrocytes when they were cocultured in vitro. MAH adhered to erythrocytes through complement receptor 1 and cell-surface sialo-glycoproteins. Importantly, MAH grew vigorously without causing any pronounced damage to erythrocytes. This erythrocyte-mediated enhancement of MAH growth occurred extracellularly depending on its direct attachment to erythrocytes. In contrast, MAH failed to multiply inside erythrocytes. Similarly, erythrocytes augmented the growth of other pathogenic mycobacteria, such as M. intracellulare and M. tuberculosis. THP-1 cell-derived human macrophages preferentially phagocytosed erythrocytes that were attached to mycobacteria (compared to bacteria alone), suggesting that erythrocyte-attached mycobacteria are an efficient infectious source for macrophages. Our findings provide new insights into the pathogenesis of mycobacterial diseases and offer an alternative and useful strategy for treating mycobacterial disease. IMPORTANCE Pathogenic mycobacteria, such as Mycobacterium tuberculosis, Mycobacterium avium subsp. hominissuis (MAH), and Mycobacterium intracellulare, cause pulmonary infections as intracellular parasites of lung macrophages and epithelial cells. Here, using histopathological examinations we found that MAH and M. intracellulare colocalized with erythrocytes in lung infection sites. Subsequent studies demonstrated that direct interaction with erythrocytes enhances the extracellular proliferation of mycobacteria based on the following results: 1. MAH adhered and invaded human erythrocytes upon coculture in vitro; 2. MAH adhered to erythrocytes through complement receptor 1 and cell-surface sialo-glycoproteins; 3. MAH rapidly proliferated when directly attached to erythrocytes but not within them; 4. other mycobacteria, such as M. intracellulare and M. tuberculosis, also proliferated in the same way as MAH. The finding that pathogenic mycobacteria grow extracellularly in an erythrocyte-dependent manner is of considerable clinical importance for understanding disease progression and latent infection.

Background: Among other negative effects, herbicides induce oxidative stress, leading to lipid peroxidation and protein oxidation. Therefore, there is a growing need to identify natural compounds with sufficient antioxidant capacity and mitigate the negative effects of herbicides without side effects. Objective: Our study aimed to examine the protective effect of the phenolic extract of wild garlic (WG) leaves on terbuthylazine-treated erythrocytes. Material and methods: In human erythrocytes treated with the herbicide terbuthylazine (4.5 mg/L) alone and a combination of terbuthylazine and WG extract, we measured malondialdehyde (MDA) and haemoglobin (Hb) concentrations and the antioxidant activities of CuZn superoxide dismutase (SOD1; EC 1.15.1.1) and catalase (CAT; EC 1.11.1.6) in vitro. Results: In comparison with terbuthylazine, WG extract reduced the concentrations of MDA and Hb from 59.69 to 43.45 nmol/gHb (27%, p < 0.001) and 165.08 to 128.64 g/L (22%, p < 0.05), respectively. Catalase activity was induced for samples treated with both WG extract and terbuthylazine compared with terbuthylazine alone (p < 0.05). Conclusions: The results demonstrated that WG may reduce the toxicity of terbuthylazine, and the erythrocyte membrane may be the primary site of phenolic action. Therefore, the lipid peroxidation intensity could be a biomarker of oxidative damage caused by terbuthylazine and the protective effect of WG. [ABSTRACT FROM AUTHOR]

Medicinal plants largely serve as a source of bioactive compounds in traditional medicines to cure various diseases. The present study was aimed at chemical composition, antioxidant, antimicrobial, cytotoxic and antihemolytic potential of five different extracts of G. hispida and H. crispum (Boraginaceae). G. hispida methanolic extract displayed highest number (eleven) of polyphenolic compounds by using high performance liquid chromatography (HPLC). Functional groups were identified by Fourier-transformed infrared spectroscopy (FTIR) and elements (Si, Fe, Ba, Mg, Ti, Ca, Mg and Cr) were observed by using laser-induced breakdown spectroscopy (LIBS) which were also highly expressed in G. hispida as compared to H. crispum. Antioxidant activity was determined via six assays and antibacterial activity was observed in decreasing order of methanol > ethanol > chloroform > ethyl acetate > n-Hexane in both species. Cytotoxic potential was investigated against brine shrimps and then liver (HepG2) and skin (HT144) cancer cell lines which was detected highest in the G. hispida ethanolic extract (50.76 % and 72.95 %). However, H. crispum chloroform extract revealed highest (31.869 μg/mL) antihemolytic activity and its methanolic extract indicated highest (13.5 %) alpha-amylase inhibitory potential. Altogether, results suggested that both species could be used effectively in food and drug industries owing to the presence of vital bioactive compounds and elements. In future, we recommend to isolate active compounds and to perform in vivo biological assays to further validate their potential biological applications. [ABSTRACT FROM AUTHOR]

Enucleated mature human erythrocytes possess NFĸBs and their upstream kinases. There is a negative correlation between eryptosis (cell death of erythrocytes) and the amount of NFĸB subunits p50 and Rel A (p65). This finding is based on the fact that young erythrocytes have the highest levels of NFĸBs and the lowest eryptosis rate, while in old erythrocytes the opposite ratio prevails. Human erythrocytes (hRBCs) effectively control the homeostasis of the cell membrane permeable anti-inflammatory signal molecule hydrogen sulfide (H2S). They endogenously produce H2S via both non-enzymic (glutathione-dependent) and enzymic processes (mercaptopyruvate sulfur transferase-dependent). They uptake H2S from diverse tissues and very effectively degrade H2S via methemoglobin (Hb-Fe3+)-catalyzed oxidation. Interestingly, a reciprocal correlation exists between the intensity of inflammatory diseases and endogenous levels of H2S. H2S deficiency has been observed in patients with diabetes, psoriasis, obesity, and chronic kidney disease (CKD). Furthermore, endogenous H2S deficiency results in impaired renal erythropoietin (EPO) production and EPO-dependent erythropoiesis. In general we can say: dynamic reciprocal interaction between tumor suppressor and oncoproteins, orchestrated and sequential activation of pro-inflammatory NFĸB heterodimers (RelA-p50) and the anti-inflammatory NFĸB-p50 homodimers for optimal inflammation response, appropriate generation, subsequent degradation of H2S etc., are prerequisites for a functioning cell and organism. Diseases arise when the fragile balance between different signaling pathways that keep each other in check is permanently disturbed. This work deals with the intact anti-inflammatory hRBCs and their role as guarantors to maintain the redox status in the physiological range, a basis for general health and well-being. [ABSTRACT FROM AUTHOR]

Flow cytometry (FC) analysis of erythrocyte shape and related biomechanical properties, such as osmotic fragility, have not moved from a research tool to regular clinical testing. The main reason is existing evidence that various pre-analytical factors influence the mathematical interpretation of the data obtained. With an aim to contribute to the standardization and broaden the use of FC for human erythrocyte shape assessment, freshly prepared peripheral blood erythrocytes isolated from healthy donors were incubated in iso and hypo-osmotic solutions (pure saline, saline with potassium and calcium, and phosphate buffered saline) and examined by FC using values of forward scatter (FSC) and side scatter (SSC). Kurtosis, skewness, Pearson's second skewness coefficient of dissymmetry (PCD), and spherical index, calculated from FSC distributions, were used for the erythrocyte shape evaluation. In all isotonic media FSC distribution and FSC-based morphology parameters showed huge inter-individual and inter-medium variation. With decreasing osmolality, in all media and samples, the size of the erythrocytes increased, and swelling index and kurtosis decreased. However, changes in skewness and PCD were influenced by the medium used and the sample tested. Compared to FSC, SSC signal in isotonic and its change in hypotonic media showed lower inter-individual variation and was not influenced by the type of medium. We propose a spherical index and kurtosis as FSC-based indicators of erythrocyte shape. As more resistant to the influence of the preanalytical treatment, SSC data appeared to be unfairly neglected for the assessment of erythrocyte shape, in comparison to the usually employed FSC data. [ABSTRACT FROM AUTHOR]

Background: Globally, lung cancer is the second most cause of deaths, which accounts for approximately 29% of the cancer cases worldwide. d-Carvone is considered a vital constituent of many essential oils with immense pharmacological benefits. Objective: In this study, we examined the immunomodulatory effect of d-carvone in the Swiss albino mice model against benzo(a)pyrene (BaP)-induced lung carcinoma. Materials and Methods: BaP was orally administered to the mice (50 mg/kg body weight [bw] for 4 weeks [twice a week]). Post-BaP administration, d-carvone (20 mg/kg bw) was orally administered to the mice (20 mg/kg bw). We calculated the tumor incidence and performed the following measurements: lung and bw, hematological parameters, immune complexes (phagocytic and avidity indexes, nitroblue tetrazolium reduction, soluble immune complex levels, immunocompetent cells (leukocytes, lymphocytes, and neutrophils), immunoglobin (Ig) levels (IgG, IgA, and IgM), level of xenobiotics and enzymes that point toward hepatic dysfunction, carcinoembryonic antigen (CEA), and proinflammatory cytokines (PICs) in experimental and normal mice. The level of oxidative stress in the experimental animals was investigated. The lung tissues of investigational animals were examined for the histopathological changes. Results: According to the results, there was an increased level of lipid peroxidation and decreased level of antioxidant activity in the lung tumor samples. The counts of lymphocytes, polymorphonuclear cells, and macrophages were notably decreased and increased, respectively, by the d-carvone post-treatment. Moreover, BaP-induced inflammation that is indicated by the increased in the level of PICs and CEA proteins in lung samples. d-Carvone attenuated the levels of PICs and CEA. Conclusion: The results of this study reveal that d-carvone prevented the cells against BaP-induced inflammation in lung cancer without causing adverse effects. [ABSTRACT FROM AUTHOR]

PURPOSE: To study the influence of human placenta extract (HPE) and its individual fractions on the thermal stability of human erythrocyte membrane. METHODS: HPE fractions were isolated by gel chromatography. Thermal hemolysis of erythrocytes, exposed to 55°C was measured spectrophotometrically. Cytosol microvscosity and barrier function of erythrocyte membranes at hyperthermia were investigated by EPR spin probe TEMPON. Thermal denaturation of erythrocyte membrane proteins were studied by differential scanning calorimetry. RESULTS: Pre-treatment of erythrocytes with HPE or its fractions inhibited thermal hemolysis. Low-molecular fractions (below 4 kDa and 12-20 kDa) were the most effective in thermal hemolysis inhibition ((31.7±3.3) % and (31.5±3.2) %, respectively). The latter fractions markedly reduced the hyperthermia (55°C)-induced permeability of erythrocytes for ferricyanide ions and inhibited the thermo-induced structural transitions in erythrocyte membrane between 40 and 50°C, which are associated with cytoskeletal proteins. HPE fractions reversibly increased the denaturation temperatures of erythrocyte membrane proteins, except that of spectrin, and enlarged the enthalpy of denaturation of all membrane proteins. CONCLUSIONS: HPE and its individual fractions increased the thermal stability of erythrocyte membranes and erythrocytes. This effect was attributed to the reversible binding of some low molecular ingredient of HPE to the integral proteins and consequent stabilization of their interaction with under-membrane cytoskeleton.

A biophysical approach was demonstrated for determining the dynamics of change in the zinc ion pool in human erythrocytes in the range from 1–1000 nM using a FluoZin-3 fluorescent probe. This method can be used in biomedical research and serve as the basis for developing diagnostic methods or predicting the course of diseases associated with a failure in zinc metabolism. [ABSTRACT FROM AUTHOR]

[Display omitted] • XAD-7 resin enhances apple pomace's phenolic compounds and bioactivity. • Purified extract inhibits cytokine expression and has anti-inflammatory activity. • Phlorizin, chlorogenic acid, and hyperoside are the main compounds in apple pomace extract. • Purified extract exhibits higher antioxidant activity compared to crude extract. • Apple extracts protected human plasma against induced oxidation and protected red blood cells. Apple pomace (AP) is a highly prevalent waste product worldwide in the fruit processing sector. This study compared the chemical profile, antioxidant, and anti-inflammatory activities of crude (CE) and an extract purified using XAD-7 resin (PE). The purification process increased the total phenolic content, flavonoids, and tannins by 3.35, 40.31, and 8.87-fold, respectively. The main phenolic compounds identified in PE were phlorizin (20.54 mg/g), chlorogenic acid (10.01 mg/g), and hyperoside (2.77 mg/g). No difference was found between CE and PE in protecting human plasma against oxidation. In human erythrocytes, both CE and PE decreased the reactive oxygen species (ROS) generation and decreased lipoperoxidation. However, PE had stronger anti-inflammatory effects than CE by promoting HO-1 gene expression, suppressing NO production, and inhibiting IL-1β , IL-6 , and IL-10 mRNA expression in lipopolysaccharide-challenged RAW.264.7 macrophages. Therefore, purifying apple pomace crude extract is a promising approach to boosting valuable antioxidants and anti-inflammatory phenolics. [ABSTRACT FROM AUTHOR]

Extreme deformability of human erythrocytes is a prerequisite for their ability to squeeze through narrow capillaries of the blood microcirculation system. Various drugs can modify this deformability and consequently provoke circulation problems. We demonstrate that microfluidic assemblies are very convenient platforms for in vitro study of the associated processes. Two types of microfluidic channels were designed to quantitatively investigate modifications of erythrocyte deformability induced by hydrogen peroxide, ethanol and pentoxifylline based on transit velocity measurements. With a high sensitivity our microfluidic assemblies show that hydrogen peroxide decreases erythrocyte deformability in a dose-dependent manner. Then, results on ethanol resolve a biphasic nature of this reactant on the deformability of single erythrocyte cells. Results on pentoxifylline provide evidence that, similar to ethanol, also this medical drug has a double-sided effect on the erythrocyte deformability, i.e. increasing the deformability at low concentrations, while decreasing it at higher ones. Taken together, our microfluidic designs propose a potent measurement method for the erythrocyte deformability, as well as providing a perspective to evaluate effects of drugs on it. • Two special microfluidics are designed to assess erythrocyte deformability changes. • Hydrogen peroxide decreases erythrocyte deformability in a dose-dependent manner. • Ethanol has a double-sided action on deformability of erythrocytes. • Pentoxifylline induces a biphasic impact on erythrocyte deformability. [ABSTRACT FROM AUTHOR]

The Red-emitting nitrogen-doped carbon dots (N-CDs) are synthesized using o-phenylenediamine by a one-step method, and can serve as a fluorescent probe for "turn off" detection of hematin in human red cells. The red-emitting N-CDs can be obtained only in acidic conditions and the emission of the red-emitting N-CDs is pH-dependent, indicating proton-controlled synthesis and emission. The red-emitting N-CDs are 2.7 nm in mean size and have a uniform dispersion and exhibit a high quantum yield (12.8%) and great optical properties. The developed sensing system for hematin displays a linear response from 0.4 to 32 μM with a detection limit of 0.18 μM. Importantly, this fluorescent probe demonstrates a good potential practicability for the quantitative detection of hematin in complex matrixes.ᅟ [ABSTRACT FROM AUTHOR]

[Display omitted] • Six novel steroidal bioconjugates linked with a 1,2,3-triazole ring were synthesized. • 1H NMR, 13C NMR, FT-IR and ESI-MS were performed for the characterization. • The in silico and in vitro study results indicate possess biological activity. • The molecular docking was performed for the selected conjugate. • Computational study was done at PM5 method level for comparative analysis. Six steroid conjugates of bile acids and sterol derivatives have been synthesized using the click chemistry method. The azide-alkyne Huisgen cycloaddition of the propionyl ester of lithocholic, deoxycholic and cholic acid with azide derivatives of cholesterol and cholestanol gave new bile acid–sterol conjugates linked with a 1,2,3-triazole ring. Previously, sterols were converted to bromoacetate substituted derivatives by reaction with bromoacetic acid bromide in anhydrous dichloromethane. These compounds were then converted to azide derivatives using sodium azide. The propiolic esters of lithocholic, deoxycholic and cholic acids were obtained by reaction with propiolic acid in the presence of p -toluenesulfonic acid. Additionally, two of these steroids: methyl 3α-propynoyloxy-12α-acetoxy-5β-cholane-24-oate and methyl 3α-propynoyloxy-7 α,12α-diacetoxy-5β-cholane-24-oate were also obtained and characterized for the first time. All conjugates were obtained in good yields using an efficient synthesis method. The structures of all conjugates and the four substrates were confirmed by spectral (1H- and 13C NMR, FT-IR) analysis, mass spectrometry (ESI-MS), and PM5 semiempirical methods. The pharmacotherapeutic potential of the synthesized compounds was estimated based on the in silico Prediction of Activity Spectra for Substances (PASS) method. The cytotoxicity of the compounds was in vitro evaluated in a hemolytic assay using human erythrocytes as a cell model. The in silico and in vitro study results indicate that the selected compound possesses an interesting biological activity and can be considered as potential drug design agent. Additionally, molecular docking was performed for the selected conjugate. [ABSTRACT FROM AUTHOR]

Propolis is a natural bee product with various beneficial biological effects. The health-promoting properties of propolis depend on its chemical composition, particularly the presence of phenolic compounds. The aim of this study was to evaluate the relationship between extraction solvent (acetone 100%, ethanol 70% and 96%) and the antifungal, antioxidant, and cytoprotective activity of the extracts obtained from propolis. Concentrations of flavonoids and phenolic acids in the propolis extracts were determined using ultrahigh-performance liquid chromatography. The antioxidant potential of different extracts was assessed on the basis of 2,2-diphenyl-1-picrylhydrazyl (DPPH·) free-radical-scavenging activity, Fe3+-reducing power, and ferrous ion (Fe2+)-chelating activity assays. The ability of the extracts to protect human red blood cell membranes against free-radical-induced damage and their antifungal activity was also determined. The results showed that the concentration of flavonoids in the propolis extracts was dependent on the solvent used in the extraction process and pinocembrin, chrysin, galangin, and coumaric acid were the most abundant phenols. All extracts exhibited high antioxidant potential and significantly protected human erythrocytes against oxidative damage. On the other hand, the antifungal activity of the propolis extracts depended on the solvent used in extraction and the fungal strains tested. It needs to be stressed that, to the best of our knowledge, there is no study relating the effect of solvent used for extraction of Polish propolis to its phenolic profile, and its antifungal, antioxidant, and cytoprotective activity.

Red blood cells (RBCs) are the most abundant cells in the human blood that have been extensively studied under morphology, ultrastructure, biochemical and molecular functions. Therefore, RBCs are excellent cell models in the study of biologically active compounds like drugs and toxins on the structure and function of the cell membrane. The aim of the present study was to explore erythrocyte ghost’s proteome to identify changes occurring under the influence of three bee venom peptides-melittin, tertiapin, and apamin. We conducted preliminary experiments on the erythrocyte ghosts incubated with these peptides at their non-hemolytic concentrations. Such preparations were analyzed using liquid chromatography coupled with tandem mass spectrometry. It was found that when higher concentrations of melittin and apamin were used, fewer proteins were identified. Moreover, the results clearly indicated that apamin demonstrates the greatest influence on the RBCs ghosts proteome. Interestingly, the data also suggest that tertiapin exerted a stabilizing effect on the erythrocyte membrane. The experiments carried out show the great potential of proteomic research in the projects focused on the toxin’s properties as membrane active agents. However, to determine the specificity of the effect of selected bee venom peptides on the erythrocyte ghosts, further proteomic research should be focused on the quantitative analysis.

Herein we describe our results on the synthesis and molecular modeling of new conjugates containing indole and uracil moiety. This group of compounds has been synthesized by the reaction of indole alkaloid - gramine with appropriate uracil (uracil, 2-thiouracil, 6-methyl-2-thiouracil, thymine, 6-methyluracil and barbituric acid) using DMF as solvent. The structures of all products were confirmed by spectroscopic ( 1 H NMR, 13 C NMR, and FT-IR) analysis, mass spectrometry (EI) and PM5 semiempirical methods. Moreover the protonation constants for investigated compounds were determined. The obtained conjugates were screened in vitro for antioxidant and haemolytic activities, as well as for the protective effects against 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH)-induced oxidative haemolysis of human erythrocytes. [ABSTRACT FROM AUTHOR]

PURPOSE: To study the influence of human placenta extract (HPE) and its individual fractions on the thermal stability of human erythrocyte membrane. METHODS: HPE fractions were isolated by gel chromatography. Thermal hemolysis of erythrocytes, exposed to 55°C was measured spectrophotometrically. Cytosol microvscosity and barrier function of erythrocyte membranes at hyperthermia were investigated by EPR spin probe TEMPON. Thermal denaturation of erythrocyte membrane proteins were studied by differential scanning calorimetry. RESULTS: Pre-treatment of erythrocytes with HPE or its fractions inhibited thermal hemolysis. Low-molecular fractions (below 4 kDa and 12-20 kDa) were the most effective in thermal hemolysis inhibition ((31.7±3.3) % and (31.5±3.2) %, respectively). The latter fractions markedly reduced the hyperthermia (55°C)-induced permeability of erythrocytes for ferricyanide ions and inhibited the thermo-induced structural transitions in erythrocyte membrane between 40 and 50°C, which are associated with cytoskeletal proteins. HPE fractions reversibly increased the denaturation temperatures of erythrocyte membrane proteins, except that of spectrin, and enlarged the enthalpy of denaturation of all membrane proteins. CONCLUSIONS: HPE and its individual fractions increased the thermal stability of erythrocyte membranes and erythrocytes. This effect was attributed to the reversible binding of some low molecular ingredient of HPE to the integral proteins and consequent stabilization of their interaction with under-membrane cytoskeleton. [ABSTRACT FROM AUTHOR]

Abstract: Carbonic anhydrase (CA) is an important metabolic enzyme family closely related to many physiological and pathological processes. Currently, carbonic anhydrase inhibitors are the target molecules in the treatment and diagnosis of many diseases. In present study, we investigated the inhibitory effects of some indazole molecules on the CA‐I and CA‐II isoenzymes isolated from human erythrocytes. We showed that human CA‐I and CA‐II activities were reduced by of some indazoles at low concentrations. IC50 values, Ki constants, and inhibition types for each indazole molecule were determined. The indazoles showed Ki constants in a range of 0.383 ± 0.021 to 2.317 ± 0.644 mM, 0.409 ± 0.083 to 3.030 ± 0.711 mM against CA‐I and CA‐II, respectively. Each indazole molecule exhibited a noncompetitive inhibition effect. Bromine‐ and chlorine‐bonded indazoles were found to be more potent inhibitory effects on carbonic anhydrase isoenzymes. In conclusion, we conclude that these results may be useful in the synthesis of carbonic anhydrase inhibitors. [ABSTRACT FROM AUTHOR]

The present study was conducted to characterise the transporter(s) responsible for the uptake of cyclic nucleotides to human erythrocytes. Western blotting showed that hRBC expressed OAT2 (SLC22A7), but detection of OAT1 (SLC22A6), or OAT3 (SLC22A8) was not possible. Intact hRBC were employed to clarify the simultaneous cyclic nucleotide egression and uptake. Both these opposing processes were studied. The Km‐values for high affinity efflux was 3.5 ± 0.1 and 39.4 ± 5.7 μM for cGMP and cAMP, respectively. The respective values for low affinity efflux were 212 ± 11 and 339 ± 42 μM. The uptake was characterised with apparently low affinity and similar Km‐values for cGMP (2.2 mM) and cAMP (0.89 mM). Using an iterative approach in order to balance uptake with efflux, the predicted real Km‐values for uptake were 100–200 μM for cGMP and 50–150 μM for cAMP. The established OAT2‐substrate indomethacin showed a competitive interaction with cyclic nucleotide uptake. Creatinine, also an OAT2 substrate, showed saturable uptake with a Km of 854 ± 98 μM. Unexpectedly, co‐incubation with cyclic nucleotides showed an uncompetitive inhibition. The observed Km‐values were 399 ± 44 and 259 ± 30 μM for creatinine, in the presence of cGMP and cAMP, respectively. Finally, the OAT1‐substrate para‐aminohippurate (PAH) showed some uptake (Km‐value of 2.0 ± 0.4 mM) but did not interact with cyclic nucleotide or indomethacin transport. [ABSTRACT FROM AUTHOR]

Although the neuronal protein α-synuclein (α-syn) is thought to play a central role in the pathogenesis of Parkinson's disease (PD), its physiological function remains unknown. It is known that α-syn is also abundantly expressed in erythrocytes. However, its role in erythrocytes is also unknown. In the present study, we investigated the localization of α-syn in human erythroblasts and erythrocytes. Protein expression of α-syn increased during terminal differentiation of erythroblasts (from day 7 to day 13), whereas its mRNA level peaked at day 11. α-syn was detected in the nucleus, and was also seen in the cytoplasm and at the plasma membrane after day 11. In erythroblasts undergoing nucleus extrusion (day 13), α-syn was detected at the periphery of the nucleus. Interestingly, we found that recombinant α-syn binds to trypsinized inside-out vesicles of erythrocytes and phosphatidylserine (PS) liposomes. The dissociation constants for binding to PS/phosphatidylcholine (PC) liposomes of N-terminally acetylated (NAc) α-syn was lower than that of non NAc α-syn. This suggests that N-terminal acetylation plays a significant functional role. The results of the present study collectively suggest that α-syn is involved in the enucleation of erythroblasts and the stabilization of erythroid membranes. [ABSTRACT FROM AUTHOR]

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A series of new di- and polyamine-caffeine analogues were synthesised and characterised by NMR, FT-IR, and MS spectroscopic methods. To access the stability of the investigated caffeine analogues, molecular dynamic simulations were performed in NAMD 2.9 assuming CHARMM36 force field. To evaluate the antioxidant capacity of new compounds, three different antioxidant assays were used, namely 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH•) scavenging activity, ferrous ions (Fe2+) chelating activity, and Fe3+→Fe2+reducing ability. In vitro, the ability of new derivatives to protect human erythrocytes against oxidative haemolysis induced by free radical from 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) was estimated. The cytotoxic activity was tested using MCF-7 breast cancer cells and human erythrocytes. All compounds showed the antioxidant capacity depending mostly on their ferrous ions chelating activity. In the presence of AAPH, some derivatives were able to effectively inhibit the oxidative haemolysis. Two derivatives, namely 8-(methyl(2-(methylamino)ethyl)-amino)caffeine and 8-(methyl(3-(methylamino)propyl)amino)caffeine, showed cytotoxic activity against MCF-7 breast cancer cells but not against human erythrocytes. Therefore, it is concluded that the selected di- and polyamine caffeine analogues, depending on their chemical structure, were able to minimise the oxidative stress and to inhibit the tumour cell growth. The confirmed antioxidant and cytotoxic properties of some caffeine derivatives make them attractive for potential applications in food or pharmaceutical industries. [ABSTRACT FROM AUTHOR]

The present investigation reports the development of PEG-modified liposomes for the delivery of naturally occurring resveratrol. PEG-modified liposomes were prepared by direct sonication of the phospholipid aqueous dispersion, in the presence of two PEG-surfactants. Small, spherical, unilamellar vesicles were produced, as demonstrated by light scattering, cryo-TEM, and SAXS. The aging of the vesicles was assessed by using the Turbiscan ® technology, and their physical stability was evaluated in vitro in simulated body fluids, results showing that the key features of the liposomes were preserved. The biocompatibility of the formulations was demonstrated in an ex vivo model of hemolysis in human erythrocytes. Further, the incorporation of resveratrol in PEG-modified liposomes did not affect its intrinsic antioxidant activity, as DPPH radical was almost completely inhibited, and the vesicles were also able to ensure an optimal protection against oxidative stress in an ex vivo human erythrocytes-based model. Therefore, the proposed PEG-modified liposomes, which were prepared by a simple and reliable method, represent an interesting approach to safely deliver resveratrol, ensuring the preservation of the carrier structural integrity in the biological fluids, and the antioxidant efficacy of the polyphenol to be exploited against oxidative stress associated with cancer. [ABSTRACT FROM AUTHOR]

Since the approval of imatinib in 2001, kinase inhibitors have revolutionized cancer therapies. Inside this family of phosphotransferases, casein kinase 2 (CK2) is of great interest and numerous scaffolds have been investigated to design CK2 inhibitors. Recently, functionalized indeno[1,2-b]indoles have been revealed to have high potency against human cancer cell lines such as MCF-7 breast carcinoma and A-427 lung carcinoma. 4-Methoxy-5-isopropyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (THN7), identified as a potent inhibitor of CK2 (IC50 = 71 nM), was selected for an encapsulation study in order to evaluate its antiproliferative activity as THN7-loaded cyclodextrin nanoparticles. Four α-cyclodextrins (α-CDs) were selected to encapsulate THN7 and all experiments indicated that the nanoencapsulation of this CK2 inhibitor in α-CDs was successful. No additional surface-active agent was used during the nanoformulation process. Nanoparticles formed between THN7 and α-C6H13 amphiphilic derivative gave the best results in terms of encapsulation rate (% of associated drug = 35%), with a stability constant (K11) of 298 mol.L-1 and a size of 132 nm. Hemolytic activity of the four α-CDs was determined before the in cellulo evaluation and the α-C6H13 derivative gave the lowest value of hemolytic potency (HC50 = 1.93 mol.L-1). Only the THN7-loaded cyclodextrin nanoparticles showing less toxicity on human erythrocytes (α-C6H13, α-C8H17 and α-C4H9) were tested against A-427 cells. All drug-loaded nanoparticles caused more cytotoxicity against A-427 cells than THN7 alone. Based on these results, the use of amphiphilic CD nanoparticles could be considered as a drug delivery system for indeno[1,2-b]indoles, allowing an optimized bioavailability and offering perspectives for the in vivo development of CK2 inhibitors. [ABSTRACT FROM AUTHOR]

The polyphenolic flavonoid quercetin has been shown to be a powerful antioxidant, in vitro and in murine models. However, its effect on redox status has been poorly examined in humans, particularly in combination with strenuous exercise. We hypothesized that quercetin supplementation would beneficially affect redox homeostasis in healthy individuals undergoing eccentric exercise. To test this hypothesis, the effects of chronic consumption of quercetin on glutathione system (reduced, oxidized, and reduced to oxidized glutathione ratio), oxidative damage [thiobarbituric acid reactive substances (TBARs)], antioxidant enzymatic network (catalase, glutathione peroxidase, superoxide dismutase) and resistance to lysis, were investigated in erythrocytes, a traditional model widely used to study the effects of oxidative stress as well as the protective effects of antioxidants. In a two weeks controlled, randomized, crossover, intervention trial, 14 individuals ingested 2 caps (1 g/d) of quercetin or placebo. Blood samples were collected before, after 2 weeks of supplementation and after a bout of eccentric exercise. Quercetin, reduced significantly erythrocytes lipid peroxidation levels and the susceptibility to hemolysis induced by the free radical generator AAPH, while no differences in antioxidant enzyme activities and glutathione homeostasis were found between the two groups. After a single bout of eccentric exercise, quercetin supplementation improved redox status as assessed by reduced/oxidized glutathione ratio analysis and reduced TBARs levels both in erythrocytes and plasma. In conclusion, our study provides evidences that chronic quercetin supplementation has antioxidant potential prior to and after a strenuous eccentric exercise thus making the erythrocytes capable to better cope with an oxidative insult. [ABSTRACT FROM AUTHOR]

Background: This study examines the effect of graduated hyperglycaemia on the state and oxygen-binding ability of hemoglobin, the correlation of phospholipid fractions and their metabolites in the membrane, the activity of proteolytic enzymes and the morphofunctional state of erythrocytes.Methods: Conformational changes in the molecule of hemoglobin were determined by Raman spectroscopy. The structure of the erythrocytes was analyzed using laser interference microscopy (LIM). To determine the activity of NADN-methemoglobinreductase, we used the P.G. Board method. The degree of glycosylation of the erythrocyte membranes was determined using a method previously described by Felkoren et al. Lipid extraction was performed using the Bligh and Dyer method. Detection of the phospholipids was performed using V. E. Vaskovsky method.Results: Conditions of hyperglycaemia are characterized by a low affinity of hemoglobin to oxygen, which is manifested as a parallel decrease in the content of hemoglobin oxyform and the growth of deoxyform, methemoglobin and membrane-bound hemoglobin. The degree of glycosylation of membrane proteins and hemoglobin is high. For example, in the case of hyperglycaemia, erythrocytic membranes reduce the content of all phospholipid fractions with a simultaneous increase in lysoforms, free fatty acids and the diacylglycerol (DAG). Step wise hyperglycaemia in incubation medium and human erythrocytes results in an increased content of peptide components and general trypsin-like activity in the cytosol, with a simultaneous decreased activity of μ-calpain and caspase 3.Conclusions: Metabolic disorders and damage of cell membranes during hyperglycaemia cause an increase in the population of echinocytes and spherocytes. The resulting disorders are accompanied with a high probability of intravascular haemolysis.