Luminol-enhanced Chemiluminescence Research Articles

Synthesis and anti-inflammatory activity of benzimidazole derivatives; an in vitro, in vivo and in silico approach

Many non-steroidal anti-inflammatory drugs (NSAIDs) concurrently inhibit both COX-1 and COX-2, with a preference for specifically targeting COX-2 due to its significant involvement in various pathologies. In addition to COX enzymes, several other targets, including Aldose reductase, Aldo-ketoreductase family 1-member C2, and Phospholipase A2, have been identified as contributors to inflammation and a myriad of other diseases.In this context, a series of 2-substituted benzimidazole derivatives was synthesized and assessed for their anti-inflammatory potential through both in vitro and in vivo assays. Molecular docking studies were conducted to elucidate the mechanism of action of these compounds against COX enzymes and other therapeutic targets associated with NSAIDs, such as Aldose reductase, AIKRC, and Phospholipase A2.Among the synthesized compounds, B2, B4, B7, and B8 demonstrated IC50 values lower than the standard ibuprofen, as determined by the Luminol-enhanced chemiluminescence assay. Validation of these findings was achieved through an in vivo carrageenan-induced mice paw edema model, confirming a comparable anti-inflammatory effect to diclofenac sodium observed in vitro. Notably, these compounds exhibited significant binding affinity with all therapeutic targets investigated in this study.These results suggest that the newly synthesized derivatives possess noteworthy anti-inflammatory potential, warranting further exploration for the development of novel multi-targeting inhibitors.

Two 5-Methoxyindole Carboxylic Acid-Derived Hydrazones of Neuropharmacological Interest: Synthesis, Crystal Structure, and Chemiluminescent Study of Radical Scavenging Properties

Given the importance of molecular structure in pharmacological activity and interaction with biological receptors, we conducted a study on the 3,4-dihydroxybenzaldehyde hydrazone derivative of 5-methoxy-indole carboxylic acid (5MICA) and a newly synthesised analogue bearing a 2-methoxy-4-hydroxyphenyl ring using single-crystal X-ray diffraction. We studied the ability of the two compounds to scavenge hypochlorite ions using luminol-enhanced chemiluminescence and their potential to modulate oxidative damage induced by iron on the biologically significant molecules lecithin and deoxyribose in order to evaluate possible antioxidant and prooxidant effects. The X-ray study revealed highly conserved geometry and limited rotation and deformation freedom of the respective indole and phenyl fragments. Interestingly, a conformational difference between the two independent molecules in the asymmetric unit of 3b was found. The X-ray study revealed a combination of hydrogen bonding interactions, short contacts, and π–π stacking stabilizing the specific three-dimensional packing of the molecules of 3a and 3b in the crystal structures. The three-dimensional packing of the molecules of 3b produced a zigzag layering projected along the c-axis. Both compounds effectively decreased luminol-dependent chemiluminescence in model systems with KO2-produced superoxide. They displayed opposite effects when applied in a xanthine/xanthine oxidase system. The hydrazones of 5MICA do not trigger a prooxidant effect or subsequent toxicity under conditions of iron-induced oxidative stress. The 3,4-dihydroxy-substituted derivative demonstrated excellent radical scavenging properties in all model systems, making it the lead compound for the development of compounds with combined neuroprotective and antioxidant properties.

Phytochemical Profiling and Therapeutic Potential of Ficus benjamina L.: Insights into Anticancer and Anti-inflammatory Activities

Background: Ficus benjamina L., commonly known as the weeping fig, is a plant traditionally used for various medicinal purposes, particularly in Southeast Asia. Despite its widespread use, there is a scarcity of scientific data validating its therapeutic properties, especially its anti-inflammatory and anticancer potentials. Objective: This study aimed to investigate the phytochemical profile of Ficus benjamina L. and to evaluate its anticancer and anti-inflammatory activities to provide a scientific basis for its traditional uses. Methods: The plant materials were collected and subjected to extraction using methanol and dichloromethane. The extracts were analyzed for primary and secondary metabolites. The anticancer activity was tested using the MTT assay on HeLa cells, and anti-inflammatory activity was assessed using a luminol-enhanced chemiluminescence method. Data analysis was conducted using IBM SPSS Statistics Version 25, focusing on inferential statistics to compare the bioactivity of the extracts against standard drugs. Results: Phytochemical analysis confirmed the presence of proteins, lipids, carbohydrates, alkaloids, flavonoids, and tannins. The dichloromethane extract showed significant anti-inflammatory activity with 58.6% inhibition at a concentration of 50 µg/ml and an IC50 of 8.966±1.03. The methanol extract exhibited minimal anticancer activity with an 8.3% inhibition rate at 30 µg/ml, suggesting low efficacy against HeLa cells compared to the standard drug, Doxorubicin, which showed a 101.2% inhibition. Conclusion: The study confirms that Ficus benjamina L. contains bioactive compounds with potential anti-inflammatory benefits and limited anticancer activity. These findings support the traditional use of the plant in treating inflammatory conditions and highlight the need for further research to optimize extraction techniques and expand biological testing.

Human Neutrophils Couple Nitric Oxide Production and Extracellular Traps Formation in Allergic Asthma.

Nitric oxide (NO) is elevated in the airways and serum of allergic asthmatic patients, suggesting an important role in asthma. NO production has been widely attributed to the canonical inducible nitric oxide synthase (iNOS). Much effort has been made to inhibit this enzyme with two outcomes: no asthma improvement; and partial NO reduction, suggesting the involvement of an iNOS-independent source. Neutrophils produce NO under inflammatory conditions and their role in asthma has been overlooked. The present study analyzes their possible role as source of NO. Our hypothesis was tested in 99 allergic patients with intermittent bronchial asthma and 26 healthy donors. NO production by blood and sputum neutrophils in response to allergens, anti-IgE, and anti-IgE receptors Abs was assessed by Griess, flow cytometry and confocal microscopy. Extracellular traps (ETs) formation, as a possible consequence of NO production, was quantified by western blot and confocal microscopy, and reactive oxygen species by luminol-enhanced chemiluminescence. Among blood and sputum granulocytes from allergic asthmatic patients, only neutrophils, produce NO by an IgE-dependent mechanism. This production is independent of NOS, but dependent on a reaction between L-arginine and reactive oxygen species from NOX2. NO and ETosis are induced in parallel, and NO amplifies ETs formation, which is a key mediator in asthma. Our findings reveal a novel role of neutrophils as the unique allergen/IgE-dependent NO source in allergic asthma enhancing ETs formation. These results suggest that NO produced by neutrophils needs further consideration in the treatment of allergic asthma.

Antioxidant, Immunomodulatory and Fungicidal Potential of Different Extracts of Raphanus Raphanistrum L. Var. Caudatus

Background: Reactive oxygen species (ROS) have been implicated in the pathogenesis of various diseases, emphasizing the importance of antioxidants in maintaining cellular homeostasis. Raphanus raphanistrum, commonly known as wild radish, has been traditionally used for its medicinal properties, including antioxidant, immunomodulatory, and antifungal effects. The plant is rich in bioactive compounds such as flavonoids, isothiocyanates, and vitamins, which contribute to its therapeutic potential. Objective: This study aimed to evaluate the antioxidant, immunomodulatory, and antifungal activities of Raphanus raphanistrum extracts and to compare these activities with standard controls. Methods: Fresh Raphanus raphanistrum plants were collected, and extracts were prepared using methanol, chloroform, and water. The antioxidant activity was assessed using the DPPH radical scavenging assay. Immunomodulatory activity was evaluated through the luminol-enhanced chemiluminescence assay in whole blood and polymorphonuclear leukocytes (PMNS), measuring the oxidative burst. The antifungal activity was determined using the agar disk diffusion method against six pathogenic fungi. The statistical analysis was performed using SPSS version 25, with results presented as mean ± standard deviation for antioxidant and antifungal assays, and mean ± standard error for immunomodulatory activity. Results: The methanol extract exhibited significant antioxidant activity with DPPH scavenging percentages ranging from 56.55% at 150 µg/ml to 87.08% at 850 µg/ml. Immunomodulatory activity showed IC50 values of 37.8 µg/ml in whole blood and 18.9 µg/ml in PMNS for the methanol extract. The antifungal activity against Candida albicans showed zones of inhibition increasing from 15 mm at 150 µg/ml to 22 mm at 850 µg/ml. These activities were compared to standard controls, including ascorbic acid for antioxidant, ibuprofen for immunomodulatory, and miconazole for antifungal assays, demonstrating the extracts' competitive potential. Conclusion: Raphanus raphanistrum extracts showed promising antioxidant, immunomodulatory, and antifungal activities, indicating their potential as natural alternatives for the prevention and treatment of related diseases. Further in vivo studies and isolation of active compounds are recommended to fully understand the mechanisms and therapeutic applications.

A rare cause of necrotizing pneumonia in an adult woman: p40phox (NCF4) deficient autosomal recessive Chronic Granulomatous Disease

IntroductionChronic Granulomatous Disease (CGD) is a rare inherited primary immunodeficiency typically diagnosed during childhood due to recurrent severe bacterial and fungal infections. Bi-allelic neutrophil cytoplasmic factor 4 (NCF4) mutations lead to P40phox deficient CGD which is a rare subtype of CGD. CaseA 37-year-old Pakistani woman with consanguineous parents presented with a left lower lobe necrotizing pneumonia. She reported tuberculosis as a child, but did not have a personal or family history of recurrent infections, colitis or other major health issues. Bronchoscopy with lung biopsy and bronchoalveolar lavage grew Burkholderia multivorans, and subsequent sputum culture grew Pseudomonas aeruginosa. Mycobacterium avium complex grew in 1 of 2 acid-fast bacillus cultures. Immune gene panel testing revealed homozygous pathologic mutations (c.118–1G > A-Splice acceptor) in NCF4 which encodes the p40phox subunit of the NADPH oxidase complex. Evaluation of her neutrophils by DHR oxidation revealed a slightly diminished response to PMA, but markedly reduced responses to multiple doses of opsonized, heat-killed S. aureus. PMN production of reactive oxygen species by luminol-enhanced chemiluminescence was also diminished after treatment with either opsonized zymosan or heat-killed S. aureus. Her neutrophils completely lacked p40phox by immunoblotting. Patient was treated with meropenem and levofloxacin. Interferon-gamma was added to her regimen following identification of her p40phox deficiency. After 10 weeks of therapy, computed tomography imaging showed improvement. Discussionp40phox is a regulatory component of the NADPH oxidase complex and impaired p40phox expression in phagocytes is associated with atypical presentations of CGD. Less than 30 cases have been described worldwide. NCF4-related CGD has fewer infections than other forms of CGD and is typically marked by granulomatous inflammation of the GI tract and skin. No adults previously identified have been asymptomatic past the second decade of life. All reported cases have had normal or only mildly impaired levels of DHR oxidation. This case report expands the clinical phenotype of NCF4-related CGD and illustrates the necessity of genetic testing for diagnosis, especially with near-normal DHR testing in the setting of opportunistic infection. Further studies are being pursued to identify why this patient presented so late in life with this infection and without colitis.

Synthesis, characterization, computational, anticancer and anti-inflammatory studies of pregabalin esters and their complexes with heavy metals

The current study entails a series of pregabalin ester complexed with zinc and copper; further characterized by spectroscopic techniques and biological assays. Docking analysis revealed three complexes with favorable binding interactions which were further subjected to in-vitro anti-inflammatory and anticancer activities. The complex-5 displayed most potent anti-inflammatory activity against Luminol-enhanced Chemiluminescence assay with IC50 value of 7.7 ± 0.8 µM as compared to standard Ibuprofen with IC50 value of 11.2 ± 1.9 µM; whereas none of them showed significant outcomes against HeLa cell lines for antiproliferative activity as compared with standard doxorubicin. Computational studies were carried out using the reported crystal structure of multidrug resistant Alpha2S protein (PDB-ID: 6ND9), neurokinin-1 receptor (PDB-ID: 2KSA) and dihydrofolate reductase (PDB-ID: 3GHW); indicating the compound 3 showed significant interactions at all receptors. As the analogues were more active than parent drug in some respects; therefore, the expansion of the project can be planned to carry out more pharmacological screening.

Exhaustive Exercise Increases Spontaneous but Not fMLP-Induced Production of Reactive Oxygen Species by Circulating Phagocytes in Amateur Sportsmen.

Simple SummaryStrenuous exercise can alter various functions of circulating phagocytes. We tested whether exhaustive exercise could influence spontaneous and fMLP-induced oxidant production (measured with luminol enhanced whole blood chemiluminescence (LBCL) normalized per phagocyte count) by blood phagocytes in amateur sportsmen. Exhaustive exercise transiently enhanced (no more than one hour from the end of bout) spontaneous oxidants generation by circulating phagocytes, while response to fMLP was decreased up to 24 h post exercise. It is suggested that a short lived increase in spontaneous oxidants production can switch on various mechanisms leading to an increase in antioxidant activity in regularly training subjects.Strenuous exercise alters the oxidative response of blood phagocytes to various agonists. However, little is known about spontaneous post exercise oxidant production by these cells. In this cross-over trial, we tested whether an exhaustive treadmill run at a speed corresponding to 70% of VO2max affects spontaneous and fMLP-provoked oxidant production by phagocytes in 18 amateur sportsmen. Blood was collected before, just after, and 1, 3, 5 and 24 h post exercise for determination of absolute and normalized per phagocyte count spontaneous (a-rLBCL, rLBCL) and fMLP-induced luminol-enhanced whole blood chemiluminescence (a-fMLP-LBCL, fMLP-LBCL). a-rLBCL and rLBCL increased by 2.5- and 1.5-times just after exercise (p < 0.05) and then returned to baseline or decreased by about 2-times at the remaining time-points, respectively. a-fMLP-LBCL increased 1.7- and 1.6-times just after and at 3 h post-exercise (p < 0.05), respectively, while fMLP-LBCL was suppressed by 1.5- to 2.3-times at 1, 3, 5 and 24 h post-exercise. No correlations were found between elevated post-exercise a-rLBCL, a-fMLP-LBCL and run distance to exhaustion. No changes of oxidants production were observed in the control arm (1 h resting instead of exercise). Exhaustive exercise decreased the blood phagocyte-specific oxidative response to fMLP while increasing transiently spontaneous oxidant generation, which could be a factor inducing secondary rise in antioxidant enzymes activity.

The Antiseptic and Antineoplastic Agent Taurolidine Modulates Key Leukocyte Functions

Although taurolidine is known to exert a wide spectrum of biological actions, its effects on immune cells have not been characterized in detail. In this study, we investigated the ex vivo effects of taurolidine on relevant innate and adaptive immune cell functions. Leukocyte functions in whole blood were assessed following treatment with various taurolidine concentrations. Viability of peripheral blood mononuclear cells (PBMCs) and granulocytes was measured using the WST-1 assay. PBMC function was assessed by measuring TNFα and IFNγ production after stimulation with lipopolysaccharide (LPS) or Candida, respectively. Reactive oxygen species (ROS) production by granulocytes was measured in whole blood using luminol-enhanced chemiluminescence. Granulocyte degranulation and activation were evaluated by membrane expression of degranulation (CD63, CD66B) and adhesion markers (CD62L, CD11b) using immunofluorescent staining followed by flow-cytometric analysis. Taurolidine decreased viability of PBMCs and granulocytes: after 2 h, IC50 concentrations were 500 and 520 μg/ml, respectively. Following prolonged exposure (≥24 h) of PBMCs, the IC50 concentrations declined to 40 μg/ml. PBMC cytokine production significantly decreased at taurolidine concentrations below the cytotoxic threshold, whereas no changes in ROS production were observed. The expression of all granulocyte adhesion and degranulation markers increased at concentrations higher than 500 μg/ml (the cytotoxic level of taurolidine). Taurolidine exhibits a dose- and time-dependent cytotoxicity toward PBMCs and granulocytes. The effects on PBMCs, as exemplified by a decrease in cytokine production, occurred below the toxic threshold, whereas granulocyte function (ROS production) remained unchanged at these taurolidine concentrations. Granulocyte activation and degranulation markers only increased at cytotoxic taurolidine concentrations.

The Differential Reactive Oxygen Species Production of Tear Neutrophils in Response to Various Stimuli In Vitro.

A large number of polymorphonuclear neutrophils (PMNs) invade the ocular surface during prolonged eye closure (sleep); these leukocytes are commonly referred as tear PMNs. PMNs contribute to homeostasis and possess an arsenal of inflammatory mediators to protect against pathogens and foreign materials. This study examined the ability of tear PMNs to generate reactive oxygen species (ROS), an essential killing mechanism for PMNs which can lead to oxidative stress and imbalance. Cells were collected after sleep from healthy participants using a gentle eye wash. ROS production in stimulated (phorbol-12-myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear PMNs was measured using luminol-enhanced chemiluminescence for 60 min. A high level of constitutive/spontaneous ROS production was observed in tear PMNs in the absence of any stimulus. While tear PMNs were able to produce ROS in response to PMA, they failed to appropriately respond to LPS and fMLP, although fMLP-stimulated tear PMNs generated ROS extracellularly in the first three minutes. Higher ROS generation was observed in isolated tear PMNs which may be due to priming from the magnetic bead cell separation system. The differential responses of tear PMNs in ROS generation provide further evidence of their potential inflammatory roles in ocular complications involving oxidative stress.

The influence of metabolic activity of platelets on sensitivity to Acetylsalicylic acid in patients with coronary heart disease

Abstract Background Acetylsalicylic acid (ASA) is used to reduce the risk of shunt occlusion after coronary artery bypass grafting (CABG). From 5% to 60% of CHD patients do not respond to ASA. This phenomenon was defined as ASA resistance. The functional activity of platelets is largely determined by the state of their metabolism. Methods The venous blood samples were acquired from 66 patients with CHD before CABG, on the first day after surgery, and on the 8–10th day after surgery. The aggregometry was carried out for all participants by an optical aggregometer with 1 mM of arachidonic acid (AA) and 5 mM Adenosinediphosphate (ADP). Resistance to ASA was determined at the level of platelet aggregation with AA over 20% on ASA therapy or over 20% after platelet incubation with ASA in vitro before CABG. Patients were divided into ASA sensitive (sASA) and ASA resistant (rASA) groups. The level of synthesis of primary and secondary reactive oxygen species (ROS) by platelets was determined using chemiluminescent analysis. We investigated the overall level of radical synthesis from the values of Imax and S (area under the chemiluminescence curve), which, respectively, characterizes the maximum synthesis per unit time and the total amount of radical. The kinetics of ROS synthesis was characterized by Tmax (time to reach the maximum for the chemiluminescent curve). The activity of NAD- and NADP-dependent dehydrogenases in platelets was determined by the bioluminescent method. Results It was found that the aggregation activity of platelets depended on the sensitivity of CHD patients to ASA and decreased during postoperative ASA therapy. The most pronounced differences in metabolic parameters of platelets in sASA and rASA patients were detected by Nox2 activity. Platelet aggregation activity was correlated with platelet Nox2 activity only in sASA patients and only before CABG. The level of AA-induced platelet aggregation with the addition of ASA in sASA patients before CABG was negatively correlated with the Imax of ADP-induced lucigenin-enhanced (r=−0.27, p=0.046) and S of spontaneous luminol-enhanced (r=−0.31, p=0.022) platelet chemiluminescence. Patients with rASA before CABG had positive correlations of AA-induced aggregation with S of spontaneous (r=0.63, p=0.029) as well as Imax (r=0.85, p&amp;lt;0.001) and S (r=0.87, p&amp;lt;0.001) of ADP-induced luminol-enhanced chemiluminescence of platelets. Therefore, the absence of correlations between platelet aggregation activity and Nox2 activity was determined by ASA resistance and postoperative ASA therapy. The synthesis of secondary ROS (Table) by platelets of CHD patients did not depend on the sensitivity of patients to ASA but increased during postoperative treatment with ASA. The activity of NAD(P)-dependent dehydrogenases in platelets did not differ in sASA and rASA patients with CHD. Conclusions Metabolic Activity of Platelets could influence on resistance to ASA in Patients with CHD. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The reported study was funded by Russian Foundation for Basic Research, Government of Krasno-yarsk Territory, Krasnoyarsk Regional Fund of Science to the research project: “Antiplatelet therapy personification in patients with coronary heart disease (CHD) depending on the level of P-selectin gene expression, the intensity of intercellular interaction and inflammation”

Neuroprotective Effect of Cinnamaldehyde on Secondary Brain Injury After Traumatic Brain Injury in a Rat Model

The aim of this study was to investigate the possible neuroprotective effects of cinnamaldehyde (CA) on secondary brain injury after traumatic brain injury (TBI) in a rat model. Rats were randomly divided into 4 groups: control (n= 9), TBI (n= 9), vehicle (0.1% Tween 80; n= 8), and CA (100 mg/kg) (n= 9). TBI was induced by the weight-drop model. In brain tissues, myeloperoxidase activity and the levels of luminol-enhanced and lucigenin-enhanced chemiluminescence were measured. Interleukin 1β, interleukin 6, tumor necrosis factor α, tumor growth factor β, caspase-3, and cleaved caspase-3 were evaluated with an enzyme-linked immunosorbent assay method. Brain injury was histopathologically graded after hematoxylin-eosin staining. Y-maze and novel object recognition tests were performed before TBI and within 24 hours of TBI. Higher myeloperoxidase activity levels in the TBI group (P < 0.001) were suppressed in the CA group (P < 0.05). Luminol-enhanced and lucigenin-enhanced chemiluminescence, which were increased in the TBI group (P < 0.001, for both), were decreased in the group that received CA treatment (P < 0.001 for both). Compared with the increased histologic damage scores in the cerebral cortex and dentate gyrus of the TBI group (P < 0.001), scores of the CA group were lower (P < 0.001). Decreased number of entries and spontaneous alternation percentage in the Y-maze test of the TBI group (P < 0.05 and P < 0.01, respectively) were not evident in the CA group. CA has shown neuroprotective effects by limiting neutrophil recruitment, suppressing reactive oxygen species and reducing histologic damage and acute hippocampal dysfunction.

The Oxidative Response of Human Monocytes to Surface Modified Commercially Pure Titanium.

Cellular responses to implanted biomaterials are key to understanding osseointegration. The aim of this investigation was to determine the in vitro priming and activation of the respiratory burst activity of monocytes in response to surface-modified titanium. Human peripheral blood monocytes of healthy blood donors were separated, then incubated with surface-modified grade 2 commercially pure titanium (CPT) disks with a range of known surface energies and surface roughness for 30- or 60-min. Secondary stimulation by phorbol 12-myrisate 13-acetate (PMA) following the priming phase, and luminol-enhanced-chemiluminescence (LCL) was used to monitor oxygen-dependent activity. Comparison among groups was made by incubation time using one-way ANOVA. One sample from each group for each phase of the experiment was viewed under scanning electron microscopy (SEM) and qualitative comparisons made. The results indicate that titanium is capable of priming peripheral blood monocytes following 60-min incubation. In contrast, 30 min incubation time lead to reduced LCL on secondary stimulation as compared to cells alone. At both time intervals, the disk with the lowest surface energy produced significantly less LCL compared to other samples. SEM examination revealed differences in surface morphology at different time points but not between differently surface-modified disks. These results are consistent with the hypothesis that the titanium surface characteristics influenced the monocyte activity, which may be important in regulating the healing response to these materials.

Taurine Chloramine and Hydrogen Peroxide as a Potential Source of Singlet Oxygen for Topical Application.

Singlet oxygen (1 O2 ) is the "active principle" in photodynamic therapy. Taurine chloramine (Tau-NHCl) and hydrogen peroxide (H2 O2 ) are well-tolerated and widely used antiseptics. Due to its mild oxidizing features and stability, Tau-NHCl can be directly used to treat skin diseases. We found that a diluted aqueous mixture of Tau-NHCl and H2 O2 acts as a slow and long-lasting potential source of 1 O2 . The reactions were studied by luminol-enhanced chemiluminescence. Evidence of the formation of 1 O2 was obtained using deuterium oxide, sodium azide and 9,10-Anthracenediyl-bis(methylene)dimalonic acid, a chemical trap of 1 O2 . The reaction was optimized, and a mechanism was proposed, including theoretical calculations at B3LYP/6-311++G(3df,2p) level of theory, adding D3Bj empirical dispersion and SMD (Water) solvent effects. Chloramines produced by the reactions between HOCl and L-alanine, 3-amino-1-propanesulfonic acid and gamma-aminobutyric acid were also prepared, and their reactivity and stability were compared with Tau-NHCl. We found that Tau-NHCl is more stable and adequate for the production of 1 O2 . In conclusion, we propose applying these drugs combination as a potential source of 1 O2 with applications for skin diseases treatment.

Evaluation of Seasonal Differences in the Antioxidant Activity of Needle Juices of Picea abies L. and Pinus sylvestris L. with Luminol-enhanced Chemiluminescence

Background: Coniferous tree species typical of the central part of Russia can be evaluated not only as sources of wood but also as a raw material for obtaining biologically active substances.Methods: A comparative evaluation of needle juices of Pinus sylvestris and Picea abies in the summer and winter periods was the objective of this study and carried out in this study by chemiluminescence analysis and testing for membrane-protective activity. Juices were evaluated for the content of flavonoids and ascorbic acid, which some authors suggest are markers of stress in plants. An increase in the content of flavonoids and ascorbic acid in Pinus sylvestris L. in the summer period is a sign of an adaptive reaction of plants to the high intensity of ultraviolet radiation, which is typical for the growing conditions of pine as a photophilous plant. An increase in these indicators in Picea abies L. in winter is a sign of the plant stress response to low temperatures. Result: The membrane-protective activity of needle juices concerning peroxidation and osmotic hemolysis was revealed, which is more prominent in Pinus sylvestris L. needle juice. The analysis revealed more prominent antioxidant properties in the juice of Pinus sylvestris L. needles compared to that of Picea abies L. both in summer and in winter.

The Role of Water in the Effect of Weak Combined Magnetic Fields on Production of Reactive Oxygen Species (ROS) by Neutrophils

Various models have been used to demonstrate the pronounced effects of the microwave frequency range electromagnetic fields, as well as weak and very weak combined collinear magnetic fields (CMF) with static and variable components in the order of micro- and nano-tesla. One of such models, previously shown to be sensitive to variations in the parameters of applied magnetic fields, is the neutrophil respiratory burst. Using luminol-enhanced cell chemiluminescence assay, we studied the effects of the CMF exerted directly on neutrophil suspensions and, indirectly, through aqueous solutions. To experimentally create a uniform standard weak CMF with 60 µT static and 100 nT alternating magnetic field components, we engineered a shielded magnetic field induction device. CMF applied directly to neutrophils enhanced reactive oxygen species (ROS) production by more than 36%. The pronounced stimulating effect was observed only when using the signals that included the frequency of 12.6 Hz that corresponds to the ion cyclotron resonance (ICR) frequency of the hydrated hydronium ion. Similarly, to direct exposure, CMF pre-treatment of a water sample subsequently added to the neutrophil suspension increased ROS production by 66%. The effect of CMF pre-treatment was retained after a series of dilutions and mechanical treatment but disappeared in “magnetic vacuum” or without mechanical influence. Therefore, weak and super weak magnetic fields may indirectly, via water, activate ROS production by neutrophils, provided that modulation of super weak component of collinear field corresponds to the ICR frequency of the hydrated hydronium ion.

The extended understanding of chronic granulomatous disease.

We briefly address the advances in genetics, pathophysiology, and phenotypes of chronic granulomatous disease (CGD). This is one of the most studied primary immunodeficiencies, which comprise mutations in genes encoding the different subunits of the NADPH oxidase system. Those mutations lead to defective reactive oxygen species production, and consequently a failure to eliminate pathogens. Patients with CGD are susceptible to fungal, bacterial, and parasitic infections. Other symptoms, as systemic adverse effects to BCG vaccine and hyperinflammation, are also important clinical conditions in this disease. This wide-ranging clinical spectrum of CGD comes from heterogeneity of mutations, X-linked-CGD or autosomal recessive inheritance, and diverse environmental pressure factors. Early accurate diagnosis and prompt treatment are necessary to diminish the consequences of the disease. The most used diagnostic tests are dihydrorhodamine, cytochrome c reduction, and luminol-enhanced chemiluminescence assay. The determination of mutations is essential for diagnosis confirmation and genetic counseling. CGD treatment usually includes prophylactic antibiotics and antifungals. Prophylactic recombinant human interferon-γ, immunosuppressors or immune modulators may be, respectively, indicated for preventing infections or inflammatory manifestations. Hematopoietic stem cell transplantation and gene therapy are currently the available options for curative treatment of CGD.

Standardized ethanol extract, essential oil and zerumbone of Zingiber zerumbet rhizome suppress phagocytic activity of human neutrophils

BackgroundZingiber zerumbet rhizome and its bioactive metabolites have previously been reported to exhibit innumerable pharmacological properties particularly anti-inflammatory activities. In the present study, the 80% ethanol extract, essential oil and zerumbone of Z. zerumbet rhizomes were explored for their in vitro immunosuppressive properties on chemotaxis, CD11b/CD18 expression, phagocytosis and chemiluminescence of isolated human polymorphonuclear neutrophils (PMNs).MethodsThe extract was analyzed quantitatively by performing a validated reversed phase high performance liquid chromatography (RP-HPLC). Zerumbone was isolated by chromatographic technique while the essential oil was acquired through hydro-distillation of the rhizomes and further analyzed by gas chromatography (GC) and GC-MS. Chemotaxis assay was assessed by using a 24-well cell migration assay kit, while CD18 integrin expression and phagocytic engulfment were measured using flow cytometry. The reactive oxygen species (ROS) production was evaluated by applying lucigenin- and luminol-enhanced chemiluminescence assays.ResultsZerumbone was found to be the most abundant compound in the extract (242.73 mg/g) and the oil (58.44%). Among the samples tested, the oil revealed the highest inhibition on cell migration with an IC50 value of 3.24 μg/mL. The extract, oil and zerumbone showed moderate inhibition of CD18 integrin expression in a dose-dependent trend. Z. zerumbet extract showed the highest inhibitory effect on phagocytic engulfment with percentage of phagocytizing cells of 55.43% for PMN. Zerumbone exhibited strong inhibitory activity on oxidative burst of zymosan- and PMA-stimulated neutrophils. Zerumbone remarkably inhibited extracellular ROS production in PMNs with an IC50 value of 17.36 μM which was comparable to that of aspirin.ConclusionThe strong inhibition on the phagocytosis of neutrophils by Z. zerumbet extract and its essential oil might be due the presence of its chemical components particularly zerumbone which was capable of impeding phagocytosis at different stages.

ROS Inhibitory Activity and Cytotoxicity Evaluation of Benzoyl, Acetyl, Alkyl Ester, and Sulfonate Ester Substituted Coumarin Derivatives.

A number of non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin, indomethacin, ibuprofen, flufenamic acid, and phenylbutazone are being clinically used to treat inflammatory disorders. These NSAIDs are associated with serious side effects such as gastric ulceration, nephrotoxicity, and bleeding. Therefore, the identification of potent and safe therapy for inflammatory disorders is still of great interest to the medicinal chemist. A series of varyingly substituted benzoyl, acetyl, alkyl ester, and sulfonate ester substituted coumarins 1-64 were screened for the inhibition of ROS, generated from zymosan activated whole blood phagocytes, using luminol-enhanced chemiluminescence technique. Among all tested compounds, 8 (IC50 = 65.0 ± 3.1 μM), 24 (IC50 = 41.8 ± 1.5 μM), 26 (IC50 = 10.6 ± 2.8 μM), 28 (IC50 = 20.9 ± 1.5 μM), and 41 (IC50 = 4.6 ± 0.3 μM) showed good anti- inflammatory potential as compared to standard antiinflammatory drug ibuprofen (IC50 = 54.3 ± 1.9 μM). Specifically, compounds 24, 26, 28, and 41 showed superior activity than standard antiinflammatory drug. Furthermore, compounds 12 (IC50 = 219.0 ± 1.4 μM), 14 (IC50 = 216.5 ± 6.2 μM), 16 (IC50 = 187.4 ± 2.2 μM), and 20 (IC50 = 196.2 ± 2.0 μM) showed moderate ROS inhibitory activity. Limited SAR study revealed that the hydroxy-substituted compound showed better ROS inhibition potential in case of 3-benzoyl and 3-ethylester coumarin derivatives. Whereas, chloro substitution was found to be important in case of 3-acetyl coumarin derivatives. Similarly, in case of sulfonate ester, chloro, and nitro groups especially at positions -4 and -3 of ring "R" played vital role in ROS inhibition. Furthermore, cytotoxicity of all active compounds was also checked on NIH-3T3 cell line. Compounds 12, 14, and 20 were found to be non-cytotoxic. Whereas, 8, 16, 24, 26, 28, and 41 were found to be very weak cytotoxic as compared to standard cycloheximide (IC50 = 0.13 ± 0.02 μM). Identified ROS inhibitors offer the possibility of additional modifications that could give rise to lead structures for further research in order to obtain more potent, and safer antiinflammatory agent.

Low-dose doxycycline inhibits hydrogen peroxide-induced oxidative stress, MMP-2 up-regulation and contractile dysfunction in human saphenous vein grafts.

Background: Cardiopulmonary bypass (CPB) applied during coronary artery bypass grafting (CABG), promotes inflammation, generation of reactive oxygen species (ROS) and up-regulation of matrix metalloproteinases (MMPs). All these complications may lead to contractile dysfunction, restenosis and early graft failure, restricting long-term efficacy of bypass grafts. Low-dose doxycycline is a potent MMP inhibitor and ROS scavenger. In this study, we aimed to investigate the effects of doxycycline on ROS generation, MMP regulation and contractile dysfunction induced by H2O2 in human saphenous vein (HSV) grafts.Methods: HSV grafts (n=7) were divided into four groups after removing endothelial layer by mechanical scratching and incubated with 10 µM H2O2 and/or 10 µM doxycycline for 16 hrs. Untreated segments served as control. Concentration–response curves to noradrenaline (NA), potassium chloride (KCl), serotonin (5-HT) and papaverine were performed. Superoxide anion and other ROS levels were determined by using lucigenin- and luminol-enhanced chemiluminescence assays, respectively. Expression/activity of gelatinases (MMP-2/MMP-9) was examined by gelatin zymography. MMP-13 expression was evaluated by immunostaining/immunoscoring.Results: H2O2 incubation increased superoxide anion and other ROS levels. Doxycycline prevented these increments. H2O2 suppressed contractile responses to NA, KCl and 5-HT. Doxycycline ameliorated contractions to NA and KCl but not to 5-HT. H2O2 or doxycycline did not altered relaxation to papaverine. MMP-2 and MMP-13 expression increased with H2O2, but doxycycline inhibited MMP-2 up-regulation/activation.Conclusion: Low-dose doxycycline may have beneficial effects on increased oxidative stress, MMP up-regulation/activation and contractile dysfunction in HSV grafts.