Xanthine Oxidase Assay Research Articles

Bioactivity-guided purification and characterization of antioxidant, anti-gout and anti-diabetic polyphenols from Panchkuta: A traditional food combination of (semi) arid regions

Desert ecosystems have thrived in part because of the contribution of local traditions, along with conventional knowledge passed down through generations. Panchkuta, a combination of five plants, including Prosopis cineraria, Cordia dichotoma, Capparis decidua, Acacia senegal, and Mangifera indica, is a classic example of ethnic desert food and how communities utilize existing food resources to ensure nutritional security. In this study, we determined the total phenolic content, total flavonoid content, antioxidant, anti-gout, and anti-diabetic activities of crude and purified (using Amberlite XAD7HP and Sephadex LH-20) fractions of Panchkuta. Further, the phenolic compounds (quercetin, tiliroside, myricetin-3-O-galactoside, and others) responsible for these activities were identified by analytical techniques, including TLC, LC-MS, and HR-MS. The purified fraction showing maximum antioxidant activity in DPPH assay (Fraction 16, IC50–156.65 ± 12.6 μg/ml) was validated for its cytoprotective and anti-lipid peroxidative effects in the in vitro cell culture system (murine fibroblast cells). Fraction 9 showed the maximum anti-gout activity in xanthine oxidase assay (IC50- 80.59 ± 9.89 μg/ml) and fraction 11 showed the maximum anti-diabetic activity in α-amylase assay (IC50 of 101.54 ± 14.7 μg/ml). The study reinforces the understanding of traditional knowledge, relevance to One Health, and authenticates the potential medicinal value of Panchkuta through scientific validation.

Unlocking the combined action of Mentha pulegium L. essential oil and Thym honey: In vitro pharmacological activities, molecular docking, and in vivo anti-inflammatory effect

Mentha pulegium L., a plant widely embraced for its therapeutic properties by populations worldwide, including Morocco, has long been recognized for its potential in treating various ailments. This study aims to comprehensively evaluate the antioxidant, anti-inflammatory, and dermatoprotective properties of essential oil derived from M. pulegium, and thyme honey as well as their combined effects. To unravel the chemical composition, a rigorous GC-MS analysis was conducted. Subsequently, we examined their antioxidant potential through three distinct assays: DPPH●, hydrogen peroxide assay, and xanthine oxidase assay. The anti-inflammatory properties were scrutinized through both in vitro and in vivo experiments. Simultaneously, the dermatoprotective efficacy was investigated in vitro by evaluating tyrosinase inhibition. Our findings revealed that pulegone constitutes the predominant compound in M. pulegium essential oil (MPEO), constituting a remarkable 74.82 % of the composition. Significantly, when the essential oil was combined with thym honey, it exhibited superior anti-inflammatory and dermatoprotective effects across all in vivo and in vitro tests. Moreover, our in silico molecular docking analysis hinted at the potential role of cyclohexanone, 3-methyl, an element found in the MPEO, in contributing to the observed outcomes. While this study has unveiled promising results regarding the combined in vitro, in vivo and in silico biological activities of the essential oil and honey, it is imperative to delve further into the underlying mechanisms through additional experimentation and alternative experimental methods. Understanding these mechanisms in greater detail will not only enhance our comprehension of the therapeutic potential but also pave the way for the development of innovative treatments and applications rooted in the synergy of these natural compounds. Furthermore, it would be advantageous to test different possible combinations using experimental design model. Moreover, it would be better to test the effect of single compounds of MPEO to clearly elucidate their efficiency. MPEO alone or combined with thyme honey may be a useful for the development of novel biopharmaceuticals.

Effects of Crocus sativus L. Floral Bio-Residues Related to Skin Protection.

The cultivation of Crocus sativus L. to obtain the saffron spice generates a large amount of biowaste, constituted mainly by the flower's tepals. The aim of this work was to evaluate the antioxidant and dermo-protective effect of a complex methanolic extract of C. sativus tepals. The extract's major phenolic content was analyzed using ultra-high performance liquid chromatography with electrospray ionization, coupled with quadrupole-time-of-flight-mass spectrometry (UHPLC-ESI-QTOF-MS). Then, the antioxidant in vitro activity of the extract was studied and related to their chemical composition. Likewise, the effect on intracellular ROS levels in HepG2 and Hs27 cell culture was determined in normal culture and under hydrogen-peroxide-induced oxidative stress. Finally, tyrosinase, hyaluronidase, collagenase, elastase, and xanthine oxidase assays were carried out to determine the dermo-protective capacity of the extract. The high polyphenol content, including flavonoids and anthocyanins, explains the antioxidant effect of the extract both in vitro and in culture assays. The extract has a significant and remarkable protective capacity against oxidative stress induced in culture of the two studied cell lines. It is also remarkable in its ability to inhibit hyaluronidase, tyrosinase, and xanthine oxidase. Results pointed out this biowaste extract as a promising ingredient in the composition of cosmetics.

Single substrate-functionalized molybdenum oxide nanozyme for specific colorimetric monitoring of xanthine oxidase activity.

Xanthine-functionalized molybdenum oxide nanodots (X-MoO3-x NDs) with peroxidase (POD)-like activity were developed for selective, sensitive, and facile colorimetric quantification of xanthine oxidase (XO). Xanthine functionalization can not only be favorable for the successful nanozyme preparation, but also for the specific recognition of XO as well as the simultaneous generation of hydrogen peroxide, which was subsequently transformed into hydroxyl radical to oxidize the chromogenic reagent based on the POD-like catalysis. Under the optimized conditions, the colorimetric biosensing platform was established for XO assay without addition of further substrates, showing good linearity relationship between absorbance difference (ΔA) and XO concentrations in the range 0.05-0.5 U/mL (R2 = 0.998) with alimit of detection (LOD) of 0.019 U/mL. The quantification of XO occursin 25 min, which is superior to the previously reported and commercial XO assays. Theproposed method has been successfully used in theassay of human serum samples, showing its highpotential in the field of clinical monitoring.

Synthesis, structural characterizations, in vitro biological evaluation and computational investigations of pyrazole derivatives as potential antidiabetic and antioxidant agents

In this study, a two pyrazole derivatives; 2-(5-methyl-1H-pyrazole-3-carbonyl)-N-phenylhydrazine-1-carboxamide (Pyz-1) and 4-amino-5-(5-methyl-1H-pyrazol-3-yl)-4H-1,2,4-triazole-3-thiol (Pyz-2) were synthesized and characterized by 13C-NMR, 1H-NMR, FT-IR, and mass spectrometry. A complete molecular structures optimization, electronic and thermodynamic properties of Pyz-1 and Pyz-2 in gas phase and aqueous solution were predicted by using hybrid B3LYP method with the 6-311++G** basis sets. Pyz-1 and Pyz-2 were evaluated in vitro for their anti-diabetic, antioxidant and xanthine oxidase inhibition activities. For anti-diabetic activity, Pyz-1 and Pyz-2 showed a potent α-glucosidase and α-amylase inhibition with IC50 values of 75.62 ± 0.56, 95.85 ± 0.92 and 119.3 ± 0.75, 120.2 ± 0.68 µM, respectively, compared to Acarbose (IC50(α-glucosidase) = 72.58 ± 0.68 µM, IC50(α-amylase) = 115.6 ± 0.574 µM). In xanthine oxidase assay, Pyz-1 and Pyz-2 exhibited remarkable inhibitory ability with IC50 values 24.32 ± 0.78 and 10.75 ± 0.54 µM, respectively. The result of antioxidant activities showed that the title compounds have considerable antioxidant and radical scavenger abilities. In addition, molecular docking simulation was used to determine the binding modes and energies between the title compounds and α-glucosidase and α-amylase enzymes.

Debittering of Emblica (Phyllanthus emblica L.) fruit powder: Preparation and biological activity

Emblica, also known as Phyllanthus emblica L., is a drug homologous food that is rich in polyphenols with various biological activities. However, its bitterness and astringency pose a significant challenge to its utilization in food products. In this study, we aimed to identify the optimal conditions for debittering Emblica. Our findings revealed that the best debittering conditions were: temperature = 50 °C, pH = 4, α-l-rhamnosidase concentration 200 U/g, and time = 5 h. High-performance liquid chromatography (HPLC) and molecular docking analysis revealed that enzymatic hydrolysis partially removed bitterness compounds. The results of antioxidant activity, xanthine oxidase, and α-glucosidase inhibitory activity assays confirmed that the Emblica fruit powder still exhibited good biological activity after enzymatic debitterization. Moreover, gastric fluids treatment might contribute to the above enhancing effect of enzymatic hydrolysates of Emblica. This study provided a theoretical basis for promoting the processing and utilization of Emblica fruit powder, as well as understanding its biological activity.

Redox potential of FAD-dependent glucose dehydrogenase

The redox potential is important to rationally employ oxidoreductases as bioelectrocatalysts to minimize electroactive interferences and maximize current density or cell voltage. For many FAD-dependent enzymes redox potentials are published, but not for FAD-dependent glucose dehydrogenase (GDH) from the glucose-methanol-choline (GMC)-oxidoreductase family, which are widely used in glucose biosensors. GDH is contacted via redox mediators or redox polymers and features a reasonably high substrate specificity and oxygen insensitivity. Here we report the redox potential of Glomerella cingulata GDH that was determined by two methods. Spectroelectrochemically we obtained a reduction potential of −0.265 ± 0.003 V vs SHE and with the xanthine oxidase assay using Janus green B a potential of −0.267 ± 0.016 V vs SHE. The determined redox potential of GcGDH differs greatly from that of Aspergillus niger glucose oxidase (−0.080 V vs SHE) despite an almost similar protein fold as GcGDH. Taking this excitingly low redox potential of GcGDH to develop optimized redox mediators and redox polymers for the fabrication of glucose oxidizing bioanodes with a low operating potential can drastically reduce the susceptibility of glucose biosensors towards electroactive substances or increase the cell voltage in biofuel cells.

Volatile composition, antidiabetic, and anti-obesity potential of Brassica incana leaf and flowering top extracts

Context Brassica incana Ten. (Brassicaceae) is an edible plant with very limited available information. Previous studies have demonstrated the polyphenolic profile and the antioxidant and cytotoxic properties of the leaf and flowering top hydroalcoholic extracts. Objective The volatile composition and the antidiabetic and anti-obesity potential of B. incana leaf and flowering top extracts have been investigated. Material and methods The volatile characterization of the extracts was attained by HS-SPME-GC/MS analysis. The antidiabetic and anti-obesity potential was investigated spectrophotometrically in vitro by the ability to modulate pancreatic lipase and α-glucosidase at different concentrations using orlistat and acarbose as reference drugs. The inhibition of advanced glycation end-products (AGEs) was measured with aminoguanidine as reference and the antioxidant activity with the xanthine/xanthine oxidase system and Trolox for comparative purposes. Results Several volatiles belonging to different chemical classes were identified, being sulphur compounds the most abundant in both leaf and flowering top extracts (56.33% and 64.40% of all volatiles). Although the leaf extract showed lower IC50 values in most of the assays (0.968 and 1.921 mg/mL for α-glucosidase; 0.192 and 0.262 mg/mL for AGEs; 0.022 and 0.038 mg/mL for superoxide scavenging), there were no statistically significant differences between both samples. These extracts showed a similar behaviour to Trolox in the xanthine oxidase assay (IC50 values of 0.022 mg/mL for leaf extract; 0.038 mg/mL for flowering top and 0.028 for Trolox). Conclusions Leaves and flowering tops from B. incana can be used as sources of functional compounds that could act as antidiabetic and anti-obesogenic agents.

Anti-Allergic and Antioxidant Potential of Polyphenol-Enriched Fractions from Cyclopia subternata (Honeybush) Produced by a Scalable Process

Anti-allergic activity was previously demonstrated for extracts of Cyclopia subternata Vogel plant material, containing substantial amounts of xanthones, benzophenones, dihydrochalcones, flavanones and flavones. Fractionation of a hot water extract on macroporous resin was performed aiming to increase its potency. Operating conditions for scaled-up fractionation of the extract were determined, using small-scale static and dynamic sorption/desorption experiments. The anti-allergic potential of the fractions was assessed based on inhibition of β-hexosaminidase release from IgE-sensitized RBL-2H3 cells. Given the role of oxidative stress in allergic reactions, the extract and fractions were also tested for their ability to scavenge the superoxide anion radical and inhibit xanthine oxidase (XO), an enzyme involved in its generation. The routine DPPH and ORAC assays were used for determination of the antioxidant capacity of the fractions. 3-β-D-Glucopyranosyl-4-O-β-D-glucopyranosyliriflophenone (IDG) had the lowest affinity for the resin, dictating selection of the optimal separation conditions. The extract was separated into four fractions on XAD1180N, using step-wise gradient elution with EtOH-water solutions. The major phenolic compounds present in the fractions were IDG and 3-β-D-glucopyranosyliriflophenone (fraction 1), mangiferin, isomangiferin, 3′,5′-di-β-D-glucopyranosyl-3-hydroxyphloretin and vicenin-2 (fraction 2), 3′,5′-di-β-D-glucopyranosylphloretin, eriocitrin and scolymoside (fraction 3) and hesperidin and p-coumaric acid (fraction 4). Fractionation was only partially effective in increasing activity compared to the extract, i.e., fractions 2, 3 and 4 in the DPPH• and XO assays, fractions 1 and 2 in the ORAC assay and fraction 1 in the β-hexosaminidase release assay. In vivo testing will be required to determine whether the increased activity of fractions is worth the effort and expense of fractionation.

Ethyl acetate partition obtained from the methanol extract of Muntingia calabura leaf exerts effective in vitro antiproliferative activity against the HT-29 colon cancer

Methanol extract of Muntingia calabura L. leaf (MEMCL) has been shown to exert the antiproliferative activity against the HT-29 (human colon adenocarcinoma) cell line. To further investigate on the medicinal potential of this plant, MEMCL was sequentially partitioned to obtain the petroleum ether, ethyl acetate and aqueous partitions, which was then tested against the HT-29 cell line and also subjected to the in vitro anti-inflammatory study. The most effective partition was also subjected to the phytoconstituents analysis using the UHPLC-ESI-MS. Findings showed that the ethyl acetate partition (EAP) exerts the most effective antiproliferative activity (IC50 = 58.0±12.9 μg/mL) without affecting the 3T3 normal fibroblast cells, exhibits the highest anti-inflammatory effect when assessed using the lipoxygenase (> 95%) and xanthine oxidase (> 70%) assays, and contained various types of polyphenolics. In conclusion, M. calabura exerts apoptotic-mediated antiproliferative activity, partly via the anti-inflammatory action and synergistic action between the polyphenolics.

Decrease of activity of antioxidant enzymes, lysozyme content, and protein degradation in milk contaminated with heavy metals (cadmium and lead)

The aim of this study was to evaluate the effect of added Cd and Pb to milk on its stability by determining antioxidant enzymatic activities, lysozyme content, and protein degradation. Antioxidant enzymatic activities were spectrophotometrically determined by superoxide dismutase, catalase, xanthine oxidase, and glutathione peroxidase assays; lysozyme was identified and quantified by HPLC-UV analysis, and protein degradation was investigated by spectrophotometric analysis of advanced oxidation protein products (AOPP) and dityrosine content. In this study, contaminated milk samples showed a significant reduction in activity of all studied enzymes compared with control milk. The contamination of milk also led to a significant reduction in the lysozyme content; lysozyme content was decreased about 22% and 18% in Pb milk and Cd milk, respectively, compared with control milk. The presence of the contaminants in the milk resulted in a significant increase of both dityrosine concentration and AOPP compared with the control milk. Moreover, between types of contaminated milk, dityrosine and AOPP values were significantly higher in the Pb milk than in the Cd milk. Therefore, it is important to monitor the presence of these toxic elements in milk for the damage they cause to consumer health both directly due to their ingestion and indirectly due to loss of milk stability.

Extracts of Andrographis paniculata (Burm.f.) Nees Leaves Exert Anti-Gout Effects by Lowering Uric Acid Levels and Reducing Monosodium Urate Crystal-Induced Inflammation.

Andrographis paniculata (Burm.f.) Nees has been found to have anti-inflammatory and immunostimulatory effects. This study was to investigate antihyperuricemic and anti-inflammatory effects of A. paniculata leaf extracts. Andrographolide, 14-deoxy-11,12-didehydroandrographolide, and neoandrographolide were quantified in 80% ethanol (EtOH80) and water extracts using High Performance Liquid Chromatography (HPLC) analysis. Antihyperuricemic activity was evaluated using a spectrophotometric in vitro inhibitory xanthine oxidase (XO) assay. The most active extract and andrographolide were further investigated in a hyperuricemic rat model induced by potassium oxonate to determine serum uric acid levels, liver XO activity, followed by Western blot analysis for renal urate transporter URAT1, GLUT9, and OAT1 to investigate the excretion of uric acid via kidney. Anti-inflammatory activity was assessed by in vitro interleukin assay for interleukin (IL-1α, IL-1β, IL-6, IL-8), and tumor necrosis factor (TNF-α) in monosodium urate (MSU) crystal-induced human fibroblast-like synoviocyte (HFLS) cells using ELISA-kits, followed by Western blot analysis for the expression of MyD88, NLRP3, NF-κB p65, and caspase-1 proteins to investigate the inflammation pathway. In vivo assay of the most active extract and andrographolide were performed based on the swelling rate and inhibition of pro-inflammatory mediator release from synovial fluid of a rat knee joint induced by MSU crystals. The results showed that the EtOH80 extract had a greater amount of andrographolide (11.34% w/w) than the water extract (1.38% w/w). In the XO inhibitory activity, none of the samples exhibited greater than 50% inhibition. However, in a rat model, EtOH80 extract (200 mg/kg/day) and andrographolide (30 mg/kg/day) decreased serum uric acid levels and reduced liver XO activity, reduced the protein expression levels of URAT1 and GLUT9, and restored the decrease in OAT1 levels. In the in vitro anti-inflammatory study, EtOH80 extract and andrographolide significantly decreased production of IL-1α, IL-1β, IL-6, and TNF-α, as well as inhibited the synthesis of MyD88, NLRP3, NF-κB p65, and caspase-1 in a concentration-dependent manner, almost comparable to dexamethasone. The EtOH80 extract (200 mg/kg/day) and andrographolide (30 mg/kg) significantly decreased swelling rate and IL-1α, IL-1β, IL-6, and TNF-α in the synovial fluid of rat models in a time-dependent manner, comparable to indomethacin (3 mg/kg/day). In conclusion, the findings show that EtOH80 extract has a substantial anti-gout effect by lowering uric acid levels and suppressing pro-inflammatory mediator production due to the andrographolide content, that might be beneficial in the treatment of gouty-inflammation.

ANTI-INFLAMMATORY AND ORAL TOXICITY ASSESSMENT OF R-38TM: A TRADITIONAL HERBAL SUPPLEMENT FOR ARTHRITIS

R-38TM is a traditional herbal supplement for treating arthritis-related conditions. High-performance liquid chromatography (HPLC) analysis was performed for identification and quantification of rosmarinic acid in the R-38TM water extract. The anti-arthritic potential of the R-38TM water extract was investigated by measuring the production of IL-6 and TNF-α in inflamed cells. Xanthine oxidase (XO) and cyclooxygenase-2 (COX-2) inhibition assays were also conducted. The cytotoxic effect of R-38TM water extract was investigated on HSF1184 cell line. Acute and subacute oral toxicity studies were conducted on female Sprague-Dawley (SD) rats. The rosmarinic acid was identified at 1.208 min (3.61 %w/w). The inflamed cells showed a decrease in the production of IL-6 (55.9%) and TNF-α (52.13%). The COX-2 and XO enzymes were moderately inhibited by R-38TM water extract. The cytotoxicity analysis showed no cytotoxic effect on the cell. The acute and subacute oral toxicity studies revealed no mortality and normal body weight at all doses. There were no significant differences (p > 0.05) in organ weight, hematological and biochemical parameters, and histology of liver and kidneys with the control group. In conclusion, R-38TM water extract exhibited no toxic effect orally and may possess potential therapeutic properties against pro-inflammatory diseases including arthritis.

The effect of chayote leaves (Sechium edule)’s flavonoid fraction on the reduction of the serum uric acid levels through the inhibition of xanthine oxidase activity

Uric acid is the final product of purine metabolism and is categorized as hyperuricemia when it reaches >6.0 mg.dL-1 for women and >7.0 mg.dL-1 for men. The chayote leaves (Sechium edule) contain a high amount of flavonoid and might be used as an alternative to reduce hyperuricemia. The purpose of this study is to analyze the effect of chayote leaves (Sechium edule)’s flavonoid fraction on the level of uric acid and the activity of xanthine oxidase (XO) in Sprague Dawley Rats. The flavonoid fraction (FF) was obtained by extracting the chayote leaves, fractionating with n-hexane, hydrolyzing with HCl, and finally re-fractionating with ethyl acetate. Thirty male Sprague Dawley rats were induced for hyperuricemia by potassium oxonate and broth block for 21 days, and the interventions were given orally for 14 days. The rats were divided randomly into five groups: normal control (K-), hyperuricemia control (K+), hyperuricemia with FF dose 50 mg.200g-1 body weight (P1), hyperuricemia with FF dose 100 mg.200g-1 body weight (P2) and hyperuricemia with allopurinol 1.8 mg.200g-1 body weight. Xanthine oxidase activity was measured by CheKineTM Xanthine Oxidase Assay Kit, with simple colorimetry methods. The statistical analysis for XO activity was done using Kruskal-Wallis followed by Mann Whitney. The results showed that chayote leaves (Sechium edule)’s flavonoid fraction contains apigenin, apigenin o-glucoside, and luteolin. It also has antioxidant activity with 98.45% inhibition. There was a significant reduction in xanthine oxidase activity in groups treated with FF (p <0.005). The best dose of FF affecting XO activity was 100 mg.200g-1 body weight. The combination of FF and allopurinol can be more effective in decreasing uric acid levels by inhibiting XO activity.

An endophytic fungus, Penicillium simplicissimum conjugated with C60 fullerene for its potential antimitotic, anti-inflammatory, anticancer and photodegradation activities

ABSTRACT In the present study, endophytic fungus, Penicillium simplicissimum isolated from Loranthus micranthus was used to analyze phytochemical studies by qualitative and GC-MS methods. The endophytic fungus P. simplicissimum yielded novel compound penisimplicissin identified through GC-MS studies. Further, P. simplicissimum was conjugated with C60 fullerene nanoparticles (Ps-FNPs) were verified using UV-vis spectra, XRD, FTIR, DLS, EDX and SEM. Ps-FNPs was confirmed using UV-visible spectra with a peak at 260 nm. The IR bands were recorded at 2085, 1428, 1181, 661, 652, 644, 628, and 604 cm-1. The Ps-FNPs treated cells showed a nucleolar shrinkage and cell arrest atprophase, binuclear and multinucleolar cells, a chromosomal bridge and diversion at anaphase was observed, whereas, chromosomal fragment and abnormal distribution at metaphase stage. The Ps-FNPs exhibited a noteworthy anticancer activity on lung cancer cell line H1975 through cytotoxicity. The cytotoxicity was induced by increasing caspase-3, 7, and 9 activities and also showed highest inhibition in xanthine oxidase and COX-II assay proved good anti-inflammatory activity. Ps-FNPs have been extensively studied for photocatalytic activity test against Rhodamine B, Methylene blue and nigrosine showed potential dye degradation in the presence of sunlight proved to be novel photocatalysts. With all the results recorded, Ps-FNPs also have a synergetic effect having on anti-mitotic, anticancer, anti-inflammation potential and photocatalytic degradation of dyes. Hence, the conjugated Ps-FNPs could be one of the potent nano-drug formulations in future. Thus, the present study gives a clear idea of the multifaceted therapeutic and photocatalytic applications.

A copper-zinc-superoxide dismutase (CuZnSOD) from redlip mullet, Liza haematocheila: Insights to its structural characteristics, immune responses, antioxidant activity, and potent antibacterial properties

Copper–zinc superoxide dismutase (CuZnSOD) is a nuclear-encoded metalloenzyme responsible for scavenging harmful reactive oxygen species (ROS). In this study, the CuZnSOD homolog from redlip mullet (Liza haematochelia) (MuCuZnSOD) was structurally and functionally characterized to evaluate its antioxidant capacity, antibacterial properties, and protective level in various pathogenic stress conditions. Structural characteristics of MuCuZnSOD were evaluated using different bioinformatics tools. Pairwise sequence comparison and evolutionary tree structure revealed that the MuCuZnSOD sequence was closely related to the CuZnSOD sequence of Oplegnathus fasciatus with a 94.2% sequence identity. Sequence alignment analysis indicated that the CuZnSOD domain was well conserved. The highest transcriptional expression of MuCuZnSOD was identified in the blood. Immune challenge with lipopolysaccharide (LPS), Lactococcus garvieae, and polyinosinic-polycytidylic acid (poly I:C) exhibited an increased MuCuZnSOD mRNA expression in the blood and liver. Transfected green fluorescent protein-fused MuCuZnSOD was localized in the cytoplasm. Recombinant MuCuZnSOD (rMuCuZnSOD) was overexpressed in a bacterial system. The rMuCuZnSOD possessed significant antioxidant properties as determined by conventional xanthine oxidase assay. The optimum pH and temperature of rMuCuZnSOD were found to be pH 9 and 25 °C, respectively. rMuCuZnSOD enzyme activity increased in a concentration-dependent manner. Treatment with potassium cyanide highly inhibited the rMuCuZnSOD activity. rMuCuZnSOD possessed a significant peroxidation activity in the presence of HCO3ˉ ions as demonstrated by the increased viability in cells treated with the enzyme in the presence of hydrogen peroxide. Antibacterial assays showed that rMuCuZnSOD had significant growth-inhibitory effects on both gram-positive and gram-negative bacteria. Collectively, our findings demonstrate that MuCuZnSOD is an essential antioxidant protein, which regulates the host defense mechanisms and innate immunity under oxidative stress.

Syntheses, Characterization and Biological Activity of Novel Thiazoylazo Dye and Its Coordination Compounds

The present work describes the synthesis of a novel heterocyclic azo dye by general diazonization of 2-amino-4,5-dimethylthiazole followed by the diazo- coupling of the resulting diazonium ion with 5-methyl-2-(propan-2-yl)phenol to obtain ligand L. This was characterized using Fourier-transformed infrared and electronic spectrophotometry. Ligand L was further coordinated with five metal ions, M:L, 1:2 [M = Cu(II), Mn(II), Zn(II), Ni(II) and Co(II)]. The coordination compounds obtained were characterized by electronic, IR spectrophotometry, magnetic susceptibility and percentage metal analyses. The results obtained suggested that a thiazoylazo dye was obtained as ligand L. It was proposed that two molecules of the solvent coordinated to the metal ion in addition with the ligands to give an octahedral geometry for copper(II), manganese(II) and nickel(II) complexes. On the other hand, square planar geometry was suggested for zinc(II) and cobalt(II) complexes. The anti-infla- mmatory activity of the ligand and coordination compounds was evaluated using four in vitro-based assays viz: xanthine oxidase and lipoxygenase inhibition assay, membrane stability and protein denaturation assay. The synthesized compounds generally exhibited good anti-inflammatory activity in all the assays carried out. However, the reference standards, in this instance, were more effective in the case of xanthine oxidase, lipoxygenase and protein denaturation inhibitory assays. For the membrane stability study, the coordination compounds and ligand L elicited more potent anti-inflammatory activity than the standard drug.

Magnesium supplementation enhances insulin sensitivity and decreases insulin resistance in diabetic rats.

Objective(s):Diabetes mellitus has been suggested to be the most common metabolic disorder associated with magnesium deficiency. This study aimed to investigate the effects and mechanisms of magnesium supplementation on insulin receptor activity in elderly type 2 diabetes using a rat model and to provide experimental evidence for insulin resistance improvement by magnesium supplementation. Materials and Methods:Rat model of type 2 diabetes was developed using a high-fat diet along with low dose streptozotocin (STZ) treatment. Magnesium supplement was given orally by mixing with the high-fat diet. Serum insulin level, insulin sensitivity, and insulin receptor affinity were assessed using radioimmunoassay (RIA). Insulin receptor, insulin receptor substrate (IRS-2), and β-Arrestin-2 gene and protein expression levels were measured using immunohistochemistry and RT-PCR. Xanthine oxidase assay, thiobarbituric acid reactive substance assay (TCA method), colorimetric assay, and ELISA were used to determine the serum SOD, MDA, T-AOC, and ox-LDL levels, respectively. Results:Magnesium supplementation enhanced insulin sensitivity and decreased insulin resistance in diabetic rats mainly through increasing insulin receptor expression, affinity, and augmenting insulin receptor signaling. Magnesium supplementation also inhibited lipid peroxidation in diabetic rats and protected against pancreatic cell injury in diabetic rats. In addition, we found that β-arrestin-2 gene expression was suppressed in diabetes, which was possibly attributed to gene methylation modification, as β-arrestin 2 promotor was rich in methylation-regulating sites. Magnesium supplementation could affect β-arrestin-2 gene expression and methylation.Conclusion:Magnesium supplementation has a positive effect on insulin receptor activity and insulin sensitivity in type 2 diabetes.

Bioproducts from forest biomass: Essential oils and hydrolates from wastes of Cupressus lusitanica Mill. and Cistus ladanifer L.

Unattended forest wastes are, among others, a potential source of wildfires, as well as a growth media for forest pests. As a way of lowering the detrimental effect of these wastes, it is important to convert these under-valued resources into a value-generating market forest wastes use. Essential oils (EOs) and hydrolates (Hs) from Cupressus lusitanica and Cistus ladanifer waste products, resulting from forest landscaping in Portugal, were evaluated for chemical composition and biological activity. Essential oils and Hs were obtained by steam-distillation (SD) and hydrodistillation (HD). Essential oils and Hs volatiles were analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC–MS). The antimicrobial activity of EOs was studied by disk agar diffusion method against Escherichia coli, Staphylococcus aureus and Candida albicans. Antioxidant activity of EOs and Hs was evaluated by 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) free radical, superoxide anion radical formation, xanthine oxidase and chelating metal ions assays. Anti-inflammatory activity of Hs was assessed by albumin denaturation assay. Monoterpene hydrocarbons and oxygen-containing monoterpenes dominated C. lusitanica EO (SD, 82–86 %, HD, 80–85 %) and Hs volatiles (SD, 93–94 %; HD 64–81 %), respectively. α-Pinene (14–36 %), limonene (8–21 %), δ-3-carene (8–19 %) and sabinene (6–18 %) were the main EO constituents. Hydrolates volatiles were dominated by cis-3-hexen-1-ol (0.1–13 %), camphor (1–11 %), umbellulone (t-48 %), p-cymene-8-ol (11–16 %) and terpinen-4-ol (21–31 %). C. ladanifer EOs were dominated by monoterpene hydrocarbons (SD, 48–80 % and HD, 29 %) and Hs by oxygen-containing monoterpenes (SD, 38–43 %, HD, 39 %). The EO major constituents were α-pinene (13–28 %) and camphene (5–25 %), whereas 2,6,6-trimethyl cyclohexanone (2–12 %) and trans-pinocarveol (5–13 %) dominated the Hs volatiles. This study reports for the first time the chemical composition of the hydrolate volatiles of these two species and their anti-inflammatory properties. Among the studied biological activities, the EOs showed the best antioxidant properties while Hs demonstrated higher anti-inflammatory activity.

Mononuclear Ni(II) Complexes with a S3O Coordination Sphere Based on a Tripodal Cysteine-Rich Ligand: pH Tuning of the Superoxide Dismutase Activity.

The superoxide dismutase (SOD) activity of mononuclear NiII complexes, whose structures are inspired by the NiSOD, has been investigated. They have been designed with a sulfur-rich pseudopeptide ligand, derived from nitrilotriacetic acid (NTA), where the three acid functions are grafted with cysteines (L3S). Two mononuclear complexes, which exist in pH-dependent proportions, have been fully characterized by a combination of spectroscopic techniques including 1H NMR, UV-vis, circular dichroism, and X-ray absorption spectroscopy, together with theoretical calculations. They display similar square-planar S3O coordination, with the three thiolates of the three cysteine moieties from L3S coordinated to the NiII ion, together with either a water molecule at physiological pH, as [NiL3S(OH2)]-, or a hydroxo ion in more basic conditions, as [NiL3S(OH)]2-. The 1H NMR study has revealed that contrary to the hydroxo ligand, the bound water molecule is labile. The cyclic voltammogram of both complexes displays an irreversible one-electron oxidation process assigned to the NiII/NiIII redox system with Epa = 0.48 and 0.31 V versus SCE for NiL3S(OH2) and NiL3S(OH), respectively. The SOD activity of both complexes has been tested. On the basis of the xanthine oxidase assay, an IC50 of about 1 μM has been measured at pH 7.4, where NiL3S(OH2) is mainly present (93% of the NiII species), while the IC50 is larger than 100 μM at pH 9.6, where NiL3S(OH) is the major species (92% of the NiII species). Interestingly, only NiL3S(OH2) displays SOD activity, suggesting that the presence of a labile ligand is required. The SOD activity has been also evaluated under catalytic conditions at pH 7.75, where the ratio between NiL3S(OH2)/ NiL3S(OH) is about (86:14), and a rate constant, kcat = 1.8 × 105 M-1 s-1, has been measured. NiL3S(OH2) is thus the first low-molecular weight, synthetic, bioinspired Ni complex that displays catalytic SOD activity in water at physiological pH, although it does not contain any N-donor ligand in its first coordination sphere, as in the NiSOD. Overall, the data show that a key structural feature is the presence of a labile ligand in the coordination sphere of the NiII ion.