Pro-oxidant Activity Research Articles

Hedera helix (Wall Ivy) leaf ethanol extract shows cytotoxic and apoptotic effects in glioblastoma cells by generating reactive oxygen species

Aim: Glioblastoma, known for its aggressiveness, accounts for most malignant gliomas and the efficiency of its treatment is still not enough showing remarkably poor prognosis even though a complex treatment approach. Hedera helix is an evergreen plant mostly known for its effects on respiratory function, especially in chronic bronchial asthma, as well as its antimicrobial, anti-inflammatory, antioxidant, and potential antitumor properties, while its effect on glioblastoma cancer and the underlying mechanism has not been elucidated yet. The aim of this study was to examine the anti-cancer properties of Hedera helix (common ivy) in relation to its ability to induce cytotoxicity, apoptosis, and reactive oxygen species production in glioblastoma (U87) cells. Material and Methods: The leaves’ ethanol, methanol, and water extracts were analyzed for phenol, flavonoid, and antioxidant levels through photometric methods. Then, different concentrations of ethanol extract were applied to U87 cells for 24 hours and analyzed for cytotoxic, apoptotic, and ROS-generating effects by measuring the luminometric intracellular ATP amount, Acridine Orange/Ethidium Bromide double staining, and DCFH-DA methods, respectively. Results: Phenol, flavonoid, and antioxidant results of all three extracts show that ethanol was best for extraction. The results showed that Hedera helix ethanol extract had dose-dependent cytotoxic and apoptotic effects with increased intracellular ROS levels. Conclusion: These results concluded that high doses of Hedera helix ethanolic extract may exhibit anti-cancer effects through pro-oxidant activity.

Sesquiterpene-evoked phytochemical toxicity in PC12 neuronal cells reveals a variable degree of oxidative stress and alpha-tocopherol and glutathione-dependent protection

Phytochemicals are often promoted generally as antioxidants and demonstrate variable levels of reactive oxygen species (ROS) sequestration in vitro, which attributes to their neuroprotective bioactivity. Sesquiterpenes from cannabis and essential oils may demonstrate bifunctional properties towards cellular oxidative stress, possessing pro-oxidant activities by generating ROS or scavenging ROS directly. Sesquiterpenes can also oxidize forming sesquiterpene oxides, however the relative contribution they make to the bioactivity or cytotoxicity of complex botanical extracts more generally is unclear, while selected cannabis-prevalent terpenes such as β-caryophyllene may also activate cannabinoid receptors as part of their biological activity. In the present study, we investigated selected sesquiterpenes β-caryophyllene and humulene and their oxidized forms (β-caryophyllene oxide and zerumbone, respectively) against established antioxidants (ascorbic acid, α-tocopherol, and glutathione) and in the presence of cannabinoid receptor 1 and cannabinoid receptor 2 antagonists, to gain a better understanding of the molecular and cellular mechanisms of neuroprotection versus neurotoxicity in semi-differentiated rat neuronal phaeochromocytoma (PC12) cells. Our results demonstrate that the sesquiterpenes β-caryophyllene, humulene and zerumbone possess concentration-dependent neurotoxic effects in PC12 cells. Both β-caryophyllene- and humulene-evoked toxicity was unaffected by CB1 or CB2 receptor antagonism, demonstrating this occurred independently of cannabinoid receptors. Both glutathione and α-tocopherol were variably able to alleviate the concentration-dependent loss of PC12 cell viability from exposure to β-caryophyllene, humulene and zerumbone. During 4-hour exposure to sesquiterpenes only modest increases in ROS levels were noted in PC12 cells, with glutathione co-incubation significantly inhibiting intracellular ROS production. However, significant increases in ROS levels in PC12 cells were demonstrated during 24-hour incubation with either antioxidants or sesquiterpenes individually, and with additive toxicity exhibited in combination. Overall, the results highlight a concentration-dependent profile of sesquiterpene neurotoxicity independent of cannabinoid receptors and dissociated from the formation of reactive oxygen species as a marker or correlate to the loss of cell viability.

Antimicrobial and Antioxidant Activity of Some Nitrogen-Containing Heterocycles and Their Acyclic Analogues.

We investigated antimicrobial and antioxidant activity of nitrogen-containing heterocycles and their acyclic analogues, some of which can be considered as promising in terms of biological activity. Based on structure, 26 tested compounds were divided into 4 groups. In the test with 2,2-diphenyl-1-picrylhydrazyl (DPPH), the compounds of the group 2 had the highest radical-binding activity (RBA) (53-78%), while those of group 3 had the lowest values (1.5-5.2%). In oxygen radical absorbance capacity assay, all compounds from groups 1, 2 and 3 showed high RBA: 44-94% at 50µM. The highest bacteriostatic activity against Escherichia coli was found for four compounds in group 2 (MIC = 0.25-1mM) and low bacteriostatic activity for group 3 (MIC > 4mM). Some relationships between the structure of compounds and the values of the MIC are revealed. It was also found that four substances from different groups had the ability to inhibit the formation of colonies in E. coli from 1.3 to 5.7 times. Four compounds reduced specific biofilm formation by 40-60%. The tested substances did not induce the expression of the sulA gene controlled by the SOS system, which indicates the lack of genotoxic activity. None of the tested compounds had pro-oxidant activity. This was shown by both the absence of production hydrogen peroxide in a bacteria-free medium and inability to induce expression of the katG gene encoding HPI catalase in growing E. coli. The data obtained could be useful in the development of new drugs.

NADPH oxidase 4 in mouse β cells participates in inflammation on chronic nutrient overload.

By exposing mice carrying a deletion of NADPH oxidase isoform 4, NOX4, specifically in pancreatic β cells (βNOX4-/-) to nutrient excess stimulated by a high-fat diet (HFD), this study aimed to elucidate the role of β-cell redox status in the development of meta-inflammation within the diabetic phenotype. The authors performed basic phenotyping of βNOX4-/- mice on HFD involving insulin and glycemic analyses, histochemistry of adipocytes, indirect calorimetry, and cytokine analyses. To characterize local inflammation, the study used caspase-1 activity assay, interleukin-1β immunochemistry, and real-time polymerase chain reaction during coculturing of β cells with macrophages. The phenotype of βNOX4-/- mice on HFD was not associated with hyperinsulinemia and hyperglycemia but showed accumulation of excessive lipids in epididymal fat and β cells. Surprisingly, mice showed significantly reduced systemic inflammation. Decreased interleukin-1β protein levels and downregulated NLRP3-inflammasome activity were observed on chronic glucose overload in βNOX4-/- isolated islets and NOX4-silenced INS1-E cells resulting in attenuated proinflammatory polarization of macrophages/monocytes in vitro and in situ and reduced local islet inflammation. Experimental evidence suggests that NOX4 pro-oxidant activity in β cells is involved in NLRP3-inflammasome activation during chronic nutrient overload and participates in local inflammatory signaling and perhaps toward peripheral tissues, contributing to a diabetic inflammatory phenotype.

Melatonin as a Repairing Agent in Cadmium- and Free Fatty Acid-Induced Lipotoxicity.

(1) Background: Cadmium (Cd) is a potentially toxic element with a long half-life in the human body (20-40 years). Cytotoxicity mechanisms of Cd include increased levels of oxidative stress and apoptotic signaling, and recent studies have suggested that these aspects of Cd toxicity contribute a role in the pathobiology of non-alcoholic fatty liver disease (NAFLD), a highly prevalent ailment associated with hepatic lipotoxicity and an increased generation of reactive oxygen species (ROS). In this study, Cd toxicity and its interplay with fatty acid (FA)-induced lipotoxicity have been studied in intestinal epithelium and liver cells; the cytoprotective function of melatonin (MLT) has been also evaluated. (2) Methods: human liver cells (HepaRG), primary murine hepatocytes and Caco-2 intestinal epithelial cells were exposed to CdCl2 before and after induction of lipotoxicity with oleic acid (OA) and/or palmitic acid (PA), and in some experiments, FA was combined with MLT (50 nM) treatment. (3) Results: CdCl2 toxicity was associated with ROS induction and reduced cell viability in both the hepatic and intestinal cells. Cd and FA synergized to induce lipid droplet formation and ROS production; the latter was higher for PA compared to OA in liver cells, resulting in a higher reduction in cell viability, especially in HepaRG and primary hepatocytes, whereas CACO-2 cells showed higher resistance to Cd/PA-induced lipotoxicity compared to liver cells. MLT showed significant protection against Cd toxicity either considered alone or combined with FFA-induced lipotoxicity in primary liver cells. (4) Conclusions: Cd and PA combine their pro-oxidant activity to induce lipotoxicity in cellular populations of the gut-liver axis. MLT can be used to lessen the synergistic effect of Cd-PA on cellular ROS formation.

Endogenous hepatic toxicity and oxidative stress in the complications of gallstones associated with cholestatic jaundice

Introduction. Cholestatic jaundice installed in gallstones is considered a multifactorial pathogenetic process. The correlation of endogenous hepatic intoxication with the reaction of the pro- and antioxidant system, characteristic of various complications of gallstones associated with cholestatic jaundice, remains an undefined subject. Material and methods. The study included the investigation of 105 patients with complicated gallstones and associated cholestatic jaundice, and the control group – 35 patients with uncomplicated gallstones. The age of the patients varied between 51 and 72 years, with an F/M ratio of 3/1. Results. The degree of endotoxemia, assessed at hospitalization by peptides with average mass values, was consistent with the inflammation present in groups 2 and 3, reporting a statistically significant difference compared to the control group [p c/2 < 0.001 (t = 11.1); p c/3 < 0.001 (t = 11.07)]. DAM, as the final product of lipid peroxidation, evaluated at hospitalization, shows double marked values in all groups compared to the control group [p < 0.001 (t = 10.7); (t = 10.9); (t = 16.5)]. The antioxidant activity, assessed at hospitalization, shows a statistically significant decrease in superoxide dismutase values [p < 0.001 (t = 6.4); (t = 4.1); (t = 5.7)], as well as catalase [p < 0.001 (t = 6.7); (t = 5.6); (t = 7.2)] in all groups, by 30-40% compared to the control group. Conclusions. In established cholestatic jaundice, there is an obvious activation of lipoperoxidation processes, contributing to the increase of endogenous intoxication and early suppression of antioxidant activity. There is a direct relationship between the increase in prooxidant activity and the level of endogenous intoxication in all complications of gallstones associated with cholestatic jaundice, but it is more elevated in infectious complications, indicating a need for an early approach.

An Effective, Green Synthesis Procedure for Obtaining Coumarin-Hydroxybenzohydrazide Derivatives and Assessment of Their Antioxidant Activity and Redox Status.

In this study, green synthesis of two derivatives of coumarin-hydroxybenzohydrazide, (E)-2,4-dioxo-3-(1-(2-(2,3,4-trihydroxybenzoyl)hydrazyl)ethylidene)-chroman-7-yl acetate (C-HB1), and (E)-2,4-dioxo-3-(1-(2-(3,4,5-trihydroxybenzoyl)hydrazyl)ethylidene)chroman-7-yl acetate (C-HB2) is reported. Using vinegar and ethanol as a catalyst and solvent, the reactions were carried out between 3-acetyl-4-hydroxy-coumarin acetate and corresponding trihydroxybenzoyl hydrazide. The antioxidant potential of these compounds was investigated using the DPPH and ABTS assays, as well as the FRAP test. The obtained results reveal that even at very low concentrations, these compounds show excellent radical scavenging potential. The IC50 values for C-HB1 and C-HB2 in relation to the DPPH radical are 6.4 and 2.5 μM, respectively, while they are 4.5 and 2.0 μM in relation to the ABTS radical. These compounds have antioxidant activity that is comparable to well-known antioxidants such as gallic acid, NDGA, and trolox. These results are in good correlation with theoretical parameters describing these reactions. Moreover, it was found that inhibition of DPPH● follows HAT, while inactivation of ABTS+● follows SET-PT and HAT mechanisms. Additionally, coumarin-hydroxybenzohydrazide derivatives induced moderate cytotoxic activity and show significant potential to modulate redox status in HCT-116 colorectal cancer cells. The cytotoxicity was achieved via their prooxidative activity and ability to induce oxidative stress in cancer cells by increasing O2˙- concentrations, indicated by increased MDA and GSH levels. Thus, ROS manipulation can be a potential target for cancer therapies by coumarins, as cancer cells possess an altered redox balance in comparison to normal cells. According to the ADMET analysis, the compounds investigated show good pharmacokinetic and toxicological profiles similar to vitamin C and gallic acid, which makes them good candidates for application in various fields of industry and medicine.

Role of Vitamin C in Targeting Cancer Stem Cells and Cellular Plasticity

Vitamin C (VC) is an essential nutrient that is vital for maintaining cellular physiology. Interestingly, it functions as either an antioxidant or a pro-oxidant, depending on the concentration used. At high-doses, VC selectively targets various cancer cell types through its pro-oxidant action, while at low-doses, VC enhances anti-tumor immunity by acting as an antioxidant. This versatility makes VC a promising anti-tumor agent for both standalone and combination therapies. Tumors consist of diverse cancer cell subtypes with distinct phenotypic and functional characteristics. In particular, cancer stem cells (CSCs), which are self-renewing multi-potent cells, are responsible for tumor recurrence, metastasis, chemoresistance, and heightened mortality. CSCs are often associated with the epithelial–mesenchymal transition (EMT), which confers increased motility and invasive capabilities that are characteristic of malignant and drug-resistant cells. Thus, eradicating CSC populations is crucial and has led to extensive efforts aimed at identifying medicines that can target them. Recent studies suggest that VC can selectively target CSCs via epigenetic and metabolic pathways in various cancers. Here, we highlight recent progress that has been made in understanding how VC effectively targets CSC evolution, providing a rationale for the use of VC either alone or in combination with other treatments to improve outcomes.

Hemolysis and Free Heme Impact Therapeutic Activity of Anti-CD20 Antibody

Hemolysis is a process of destruction of erythrocytes that can occur in different hereditary or acquired pathologies. It is accompanied by release of hemoglobin and heme, which both manifest potent pro-oxidative and pro-inflammatory activities. If released in large quantities hemoglobin and heme can directly contribute to systemic inflammation and tissue injury in hemolytic diseases. Moreover, it was demonstrated that heme binds to a significant fraction (ca. 10 %) of human antibodies (Abs) and this binding can result in considerable functional changes. Thus, heme-bound human IgG acquire reactivity towards a large set of unrelated proteins, including autoantigens. In a previous study we observed that 14 % of a repertoire of 117 clinical-stage therapeutic mAbs are able to bind heme and as a result they acquired autoreactivity and polyreactivity. Among these Abs some are specific for CD20 and used in lymphoma therapy (e.g. Rituximab). Importantly, therapeutic mAbs can be administered in patients with ongoing hemolysis. Thus, it is of critical importance to decipher molecular mechanism and functional consequences of interaction of heme with therapeutic mAbs and understand how the therapy of lymphoma can be affected by underling hemolysis and heme release. The present study has the following objective: to elucidate the effect of heme on the therapeutic activity of anti-CD20 therapy in in vitro and in vivo models of B cell lymphoma. Effect of heme on anti-CD20 therapeutic Abs was assessed by i) binding assays - ELISA, western blot; protein microarrays; absorbance spectroscopy; surface plasmon resonance, and ii) molecular modeling. The impact of heme on functional and therapeutic activity of Rituximab was analyzed by i) site-directed mutagenesis; ii) flow cytometry; iv) in vitro and in vivo models of human B cell lymphoma. Our data reveled molecular and functional details about the interaction of heme with Rituximab and other anti-CD20 Abs. Thus, we characterized the interaction of Abs with heme, and identified in case of Rituximab the putative binding site for heme that overlapped with antigen binding site. By using a protein array with >9800 human proteins, we identified the target proteins of heme-bound Rituximab. A mouse model of human B cell lymphoma revealed that the therapeutic activity of Rituximab was compromised following exposure to cell free heme under condition of acute intravascular hemolysis. Our study delineates that a fraction of currently used therapeutic mAbs show high sensitivity to heme and their therapeutic activity can be compromised under hemolytic conditions. This finding suggests that therapeutic interventions that reduce free heme and /or intravascular hemolysis should precede therapy with these Abs. Moreover, we identified sequence features of Abs that determine the sensitivity to heme. This finding can help to reduce sensitivity of heme of therapeutic Abs by protein engineering.

The role of dysmetabolic iron overload syndrome in non-alcoholic fatty liver disease and carbohydrate metabolism disorders induction

Iron affects the pathogenesis and clinical course of several chronic metabolic diseases such as obesity, atherosclerosis, non-alcoholic fatty liver disease and type 2 diabetes mellitus. High pro-oxidant iron activity is physiologically controlled by mechanisms regulating entry, recycling, and loss of body iron. These mechanisms include the interplay of iron with ferritin, transferrin, hepcidin, insulin, as well as with adipokines and proinflammatory molecules. An imbalance of these regulatory mechanisms results in both systemic and parenchymal siderosis. Iron overload has a toxic effect on the major tissues involved in lipid and glucose metabolism — pancreatic β cells, liver, muscle, and adipose tissue — as well as the organs affected by chronic hyperglycemia — brain, retina and kidneys. Hyperferremia leads to a decrease in insulin secretion, the formation of insulin resistance and increased liver gluconeogenesis. Molecular mechanisms for these effects are diverse. Elucidating them will implicate both for carbohydrate metabolism disorders prevention and for the pathogenesis of other diseases that are, like diabetes mellitus type 2, associated with nutrition, aging and iron. The literature review presents data from world studies on the mutual influence of glucose metabolism and iron overload, and discusses the differences between hereditary and acquired disorders of iron metabolism from the standpoint of their influence on carbohydrate metabolism.

Redox-Catalytic Properties of Cobalamins

Vitamin B12, or cobalamin, is essential for normal body function and used in the therapy of different diseases. A several studies have shown that vitamin B12 has anti-inflammatory and antioxidant properties that can play an important role in the prevention of some diseases. On the other hand, it has been reported that vitamin B12 in combination with such reducing agents as ascorbate (vitamin C) and thiols showed prooxidant activity. This review provides information on the roles of vitamin B12 in diseases accompanied by inflammation and oxidative stress and the effects of vitamin B12 administrated alone and in combinations with different reducing agents such as ascorbate and thiols on oxidative stress. In addition, the mechanisms of prooxidant actions of combinations of vitamin B12 with these reducing agents depending on the form of vitamin B12 (hydroxocobalamin and cyanocobalamin) are discussed. Understanding the mechanisms of prooxidant action of vitamin B12 is necessary for developing strategies for therapeutic administration of vitamin B12.

Role of Vitamins in Cardiovascular Health: Know Your Facts - Part 1.

Cardiovascular (CV) disease (CVD) is a major cause of morbidity and mortality world-wide, thus it is important to adopt preventive interventions. Observational data demonstrating CV benefits of vitamin supplements, advanced by self-proclaimed experts have resulted in ~50% of Americans reporting the use of multivitamins for health promotion; this practice has led to a multi-billion-dollar business of the multivitamin-industry. However, the data on the extensive use of multivitamins show no consistent benefit for CVD prevention or all-cause mortality, while the use of certain vitamins might prove harmful. Thus, the focus of this two-part review is on the attributes or concerns about specific vitamins on CVD. In Part 1, the CV effects of specific vitamins are discussed, indicating the need for further supportive evidence of potential benefits. Vitamin A preserves CV homeostasis as it participates in many biologic functions, including atherosclerosis. However, supplementation could potentially be harmful. Betacarotene, a pro-vitamin A, conveys pro-oxidant actions that may mitigate any other benefits. Folic acid alone and certain B-vitamins (e.g., B1/B2/B6/B12) may reduce CVD, heart failure, and/or stroke, while niacin might increase mortality. Vitamin C has antioxidant and cardioprotective effects. Vitamin D may confer CV protection, but all the data are not in agreement. Combined vitamin E and C have antiatherogenic effects but clinical evidence is inconsistent. Vitamin K seems neutral. Thus, there are individual vitamin actions with favorable CV impact (certain B-vitamins and vitamins C and D), but other vitamins (β-carotene, niacin) may potentially have deleterious effects, which also holds true for high doses of fat-soluble vitamins (A/D/E/K).

Evaluation of the generation of reactive oxygen species and antibacterial activity of honey as a function of its phenolic and mineral composition.

The antibacterial activity (ABA) of honey is associated with the generation of reactive oxygen species (ROS), where polyphenols (PFs) play a key role due to their pro-oxidant action modulated by metallic cations. In this work, the contents of PFs, H2O2, OH radicals, Cu, Fe, Mn, Zn, and ABA against Staphylococcus epidermidis and Pseudomonas aeruginosa were determined in honeys from central Chile. Then, their relationships were evaluated through partial least squares regression. The average contents of phenolic acids, flavonoids and metals in honey ranged from 0.4 to 4μg/g, 0.3-1.5μg/g and 3-6μg/g, respectively. All honeys showed accumulation of H2O2 (1-35μg/g) and OH radicals. The PLS showed that gallic acid, p-coumaric acid, chrysin, kaempferol, Fe, and Mn stimulate the generation of ROS. Quercetin, Cu, and Zn showed marginal antioxidant effects. PFs favor the ABA of honey against both bacteria and H2O2 against S. epidermidis.

A Nutraceutical Compound Containing a Low Dose of Monacolin K, Polymethoxyflavones, Phenolic Acids, Flavonoids, and Hydroxytyrosol Improves HDL Functionality.

In earlier studies, it has been observed that 8-week treatment with a novel nutraceutical compound (NC) containing low monacolin K dose, polymethoxyflavones, phenolic acids, flavonoids, and hydroxytyrosol improves lipid profile and endothelial function and reduces the level of oxidized low-density lipoprotein (oxLDL). We hypothesize that this effect might be, at least in part, explained by positive modulation exerted by the NC on the atheroprotective function of high-density lipoprotein (HDL). This study aimed to evaluate whether the NC could influence determinants of HDL function. Forty-five subjects with low-moderate dyslipidaemia were enrolled and treated for 8 weeks with the NC, followed by 4 weeks of washout. Blood samples were collected at every time point to evaluate changes in lipid profile, endothelial function, oxLDL, and markers of HDL function, such as the anti-oxidant activities of paraoxonase-1, glutathione peroxidase-3 (Gpx3), lipoprotein-phospholipase A2 (Lp-PLA2), and pro-oxidant activity of myeloperoxidase (MPO). Although the concentration of HDL-C did not change, the activity of Lp-PLA2 significantly decreased upon treatment (-11.6%, p<0.001) and returned to baseline level 4 weeks after the end of treatment. In contrast, Gpx3 increased after treatment (+5%, p<0.01) and remained unvaried after 4 weeks. Both MPO activity and concentration significantly decreased after the washout period (-33 and 32%, p<0.001). For the first time, it was found that the administration of an NC with beneficial effects on lipid homeostasis also positively impacts HDL function by improving the balance between protective and damaging determinants. Further investigation is required to corroborate our findings.

Anti-tumor activity of silymarin nanoliposomes in combination with iron: In vitro and in vivo study

Combination therapy represents a promising strategy in cancer management by reducing chemotherapy resistance and associated side effects. Silymarin (SLM) has been extensively investigated due to its potent antioxidant properties and demonstrated efficacy against cancer cells. Under certain conditions however, polyphenolic compounds may also exhibit prooxidant activity by elevating intracellular reactive oxygen species (ROS), which can harm the target cells. In this study, we hypothesized that the simultaneous administration of iron (Fe) could alter the antioxidant characteristic of SLM nanoliposomes (SLM Lip) to a prooxidant state. Hence, we first developed a SLM Lip preparation using lipid film method, and then investigated the anti-oxidant properties as well as the cytotoxicity of the liposomal preparation. We also explored the efficacy of concomitant administration of iron sucrose and SML Lip on the tumor growth and survival of mice bearing tumors. We observed that exposing cells to iron, and consecutive treatment with SLM Lip (Fe + SLM Lip) could induce greater toxicity to 4 T1 breast cancer cells compared to SLM Lip. Further, Fe + SLM Lip combination demonstrated a time-dependent effect on reducing the catalase activity compared to SLM Lip, while iron treatment did not alter cell toxicity and catalase activity. In a mouse breast cancer model, the therapeutic efficacy of Fe + SLM Lip was superior compared to SLM Lip, and the treated animals survived longer. The histopathological findings did not reveal a significant damage to the major organs, whereas the most significant tumor necrosis was evident with Fe + SLM Lip treatment. The outcomes of the present investigation unequivocally underscored the prospective use of Fe + SLM combination in the context of cancer therapy, which warrants further scrutiny.

Theoretical Insights into the Oxidative Stress-Relieving Properties of Pinocembrin─An Isolated Flavonoid from Honey and Propolis.

Bee products are a valuable group of substances that have a wide range of applications for humans. They contain a high level of polyphenolic compounds, which have been shown to combat radicals and effectively reduce oxidative stress. In this study, density functional theory was utilized to determine the anti-OOH activity, sequestration of free Cu(II) and Fe(III) ions, the potential pro-oxidative activity of the formed complexes, and the repairing capabilities toward essential biomolecules. The kinetic constants for scavenging of hydroperoxide radical were found to be low, with an order of magnitude not exceeding 10-3 M-1 s-1. Chelating properties showed slightly more satisfactory outcomes, although most complexes exhibited pro-oxidant activity. Pinocembrin, however, proved effective in repairing oxidatively damaged biological compounds and restoring their original functionality. The study found that whilst the system displays limited type I and type II antioxidant activity, it may support the role of physiological reductants already present in the biological matrix.

Sperm Quality Assessment in Stallions: How to Choose Relevant Assays to Answer Clinical Questions.

Stallion sperm analysis is indicated for infertility diagnosis, pre-sale expertise, production of fresh or frozen doses, and frozen straw quality control. Various collection methods are described, and numerous assays can be performed on semen. Determining an approach for each of these cases is challenging. This review aims to discuss how to obtain relevant clinical results, answering stallion owners' concerns. Semen can be collected with an artificial vagina on a phantom or a mare, by electro-ejaculation under anesthesia, or after pharmacological induction. The collection method influences the semen volume and concentration, while the total sperm number depends on the testicular production and collection frequency. In the seminal plasma, acidity, pro-oxidant activity, and some enzymes have repercussions for the semen quality and its conservation. Moreover, non-sperm cells of seminal plasma may impact semen conservation. Motility analysis remains a core parameter, as it is associated with fresh or frozen dose fertility. Computer-assisted motility analyzers have improved repeatability, but the reproducibility between laboratories depends on the settings that are used. Morphology analysis showing spermatozoa defects is useful to understand production and maturation abnormalities. Staining of the spermatozoa is used to evaluate viability, but recent advances in flow cytometry and in fluorochromes enable an evaluation of multiple intracellular parameters. Spermatozoa protein expression already has clinical applications, for example, as a fertility and freezing ability predictor. At present, stallion semen analysis ranges from macroscopic evaluation to assessing spermatozoa proteins. However, clinically, all these data may not be relevant, and the lack of standardization may complicate their interpretation.

CHOLINESTERASE INHIBITION POTENTIAL OF ENDEMIC SATUREJA SUBSPICATAL.ESSENTIAL OIL AND EXTRACTS

Medicinal plants are potentialsources of bioactive compounds.One of the medicinal plants used in the traditional medicine of Bosnia and Herzegovina isendemic Satureja subspicataL. In this work, we examined the ability of Satureja subspicataL. essential oil and hot water and methanol extractsto inhibit the enzymes acetylcholinesterase(AChE) and butyrylcholinesterase(BChE) using Ellman’s method.The ability ofSatureja subspicataL. essential oil in concentration of 1 mg/mL and 2 mg/mL to inhibit enzymes was moderate: 72.82%, and 76.89% for AChE, and 51.51%, and 27.15% for BChE, respectively. Analyzed hot water and methanol extractsin concentration of 1 mg/mL showed weak ability of cholinesterase inhibition. Extracts were additionally analyzed regardingtoability to protect proteins from oxidation, during 1 h and 24 h incubation periods. After incubation for 1 hhot water extractshowed a very good protective effect(10.61%), while the methanolic extract showed prooxidative activity. After incubation for 24 h, both extracts showed prooxidative activity.The obtained results show that the examined essential oil and extracts of S. subspicataL. containcompounds withcholinesterase inhibition and antioxidant potential, and thereforecan be useful in treatment of Alzheimer's disease.KEYWORDS:Satureja subspicata, essential oil, extracts, cholinesterase inhibition, protein oxidation

Simultaneously Determined Antioxidant and Pro-Oxidant Activity of Randomly Selected Plant Secondary Metabolites and Plant Extracts.

Oxidative stress is a well-known phenomenon arising from physiological and nonphysiological factors, defined by the balance between antioxidants and pro-oxidants. While the presence and uptake of antioxidants are crucial, the pro-oxidant effects have not received sufficient research attention. Several methods for assessing pro-oxidant activity, utilizing various mechanisms, have been published. In this paper, we introduce a methodology for the simultaneous determination of antioxidant and pro-oxidant activity on a single microplate in situ, assuming that the FRAP method can measure both antioxidant and pro-oxidant activity due to the generation of pro-oxidant Fe2+ ions in the Fenton reaction. Systematic research using this rapid screening method may help to distinguish between compounds with dominant antioxidant efficacy and those with dominant pro-oxidant effects. Our preliminary study has revealed a dominant pro-oxidant effect for compounds with a higher number of oxygen heteroatoms, especially sp2 hybridized compounds (such as those containing keto groups), such as flavonoids and plant extracts rich in these structural types. Conversely, catechins, carotenoids, and surprisingly, extracts from birch leaves and chestnut leaves have demonstrated dominant antioxidant activity over pro-oxidant. These initial findings have sparked significant interest in the systematic evaluation of a more extensive collection of compounds and plant extracts using the developed method.

In vitro evaluation of antiatherogenic potential of Origanum × paniculatum, Lippia alba, Clinopodium nepeta, and Eucalyptus globulus essential oils

IntroductionEssential oils (EOs) are mixtures of secondary plant metabolism compounds with potential lipid-lowering activity and antioxidant properties. They may therefore be considered as add-on therapy for the prevention and treatment of cardiovascular diseases. MethodsEOs of Origanum × paniculatum (OpEO), Lippia alba (chemotype linalool) (LaLEO), Clinopodium nepeta (CnEO) and Eucalyptus globulus (EgEO), were evaluated on THP-1 foam cell macrophages as a cellular model of atherogenesis. Cell viability was determined by the MTT assay, lipid content by thin-layer chromatography, and Oil Red O staining. Antioxidant activity was evaluated by TBARS on low-density lipoprotein lipid peroxidation, and by ABTS•+, DPPH•, and FRAP assays. EOs were characterised by gas chromatography–mass spectrometry and a predictive multivariate data analysis was performed by partial least square regression (PLS) to identify the components related to antiatherogenic potential. ResultsOpEO reduced foam cell viability (IC50 = 45 µl/l) and cholesterol and triacylglycerol content and demonstrated antioxidant activity. In contrast, LaLEO increased lipid content and showed a prooxidant capacity with low reduction of cell viability (IC50 = 340 µl/l). CnEO showed prooxidant activity and reduction of cell viability (IC50 = 130 µl/l) without changes in lipid content. EgEO showed antioxidant activity, lipid increase, and low cytotoxicity (IC50 = 770 µl/l). PLS analysis suggests that cis-sabinene hydrate is responsible for cell viability inhibition, thymol and β-cis-terpineol decrease lipid content and possess antioxidant activity, and linalool increases lipid levels and has prooxidant capacity. ConclusionsOpEO is a promising natural product for therapy against atherosclerosis, with cis-sabinene hydrate, thymol, and β-cis-terpineol as potential active principles.