Study on drug-target binding kinetics profiles of flavonoids in Chrysanthemum morifolium and xanthine oxidase / 中国中药杂志

Based on the target occupancy mathematical model, the binding kinetic process of potential active ingredients of lowering uric acid in Chrysanthemum morifolium with xanthine oxidase(XOD) was evaluated. The potential active ingredients of lowering uric acid in Ch. morifolium were screened by UPLC-Q-Exactivems MS technology, reference substance identification and in vitro enzymatic kinetics experiments. The binding kinetic parameters of xanthine oxidase and potential inhibitor in Ch. morifolium were determined by surface plasma resonance(SPR). The verified mathematical model of the XOD target occupancy evaluated the kinetic binding process of inhibitors and xanthine oxidase in vivo. According to UPLC-Q-Exactive MS and reference substance identification, 39 potential uric acid-lowering active ingredients in Ch. morifolium extracts were identified and the inhibitory activities of 23 compounds were determined. Three potential xanthine oxidase inhibitors were screened, namely genistein, luteolin, and apigenin. whose IC_(50 )were 1.23, 1.47 and 1.59 μmol·L~(-1), respectively. And the binding rate constants(K_(on)) were 1.26×10~6, 5.23×10~5 and 6.36×10~5 mol·L~(-1)·s~(-1), respectively. The dissociation rate constants(K_(off)) were 10.93×10~(-2), 1.59×10~(-2), and 5.3×10~(-2 )s~(-1), respectively. After evaluation by different administration methods, the three selected compounds can perform rapid and sustained inhibition of xanthine oxidase in vivo under combined administration. This study comprehensively evaluated the target occupancy process of three effective components in different ways of administration in vivo by UPLC-MS, concentration-response method, SPR technology and xanthine oxidase target occupancy model, which would provide a new research idea and method for screening active ingredients in traditional Chinese medicine.

Xanthine oxidase activity in patients with age-related cataract associated with hypertension

Reactive oxygen species and lipid peroxidation are important factors that contribute to the development of age-related cataract. The study included 130 patients with age-related cataract, 69 of whom were diagnosed with hypertension (HT), 20 with hypertension and type 2 diabetes mellitus (DM), and 41 had no accompanying condition. The following parameters were measured in the serum of the examinees: products of lipid peroxidation malondialdehyde (MDA) and lipofuscin-like fluorophores (LLF), activity of prooxidative enzymes xanthine oxidase (XO) and myeloperoxidase (MPO), antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), the concentration of thiol groups, and the ferric reducing activity of plasma. The activity of prooxidative enzymes XO and MPO was higher in the plasma of patients with HT (XO=9.0±1.2 U/L; MPO=77.3±8.4 U/L) and with HT and DM (XO=11.9±0.9 U/L; MPO=89.5±5.0 U/L) compared to patients with age-related cataract (XO=6.2±0.9 U/L; MPO=52.4±6.3 U/L; P<0.01). Our research has shown that patients with age-related cataract and hypertension were exposed to increased oxidative damage of biomolecules, based on the increased plasma LLF and MDA content and decreased levels of thiol groups. Oxidative changes of biomolecules in these patients were associated with increased activity of the XO, MPO, and GPx enzymes and a lower extracellular SOD activity and total ferric reductive ability of plasma.

L-Arginine attenuates oxidative stress condition during cardiomyopathy.

Increased production of oxygen free radicals and decreased oxidant capacity occur in coronary artery diseases (CAD). This pro-oxidant shift in intracellular redox state may induce cell death by either direct cell membrane damage by lipid peroxidation or apoptosis through activation of transcription factors. These changes occur not only in cardiomyocytes, but also in cardiac sympathetic nerves, which are very sensitive to oxidative damage. Patients with heart failure encounter reduced peripheral blood flow at rest, during exercise and in response to endothelium-dependent vasodilators. Current treatments of cardiomyopathy, a degenerative condition of the myocardium frequently associated with heart failure have done little to enhance patient survival. Decreased myocardial contractility and altered regulation of peripheral circulation along with oxidative conditions are important contributors to the symptoms and prognosis of the disease process. Nitric oxide formed from L-arginine (2-amino-5 guanidinovaleric acid) metabolism in endothelial cells contributes to regulation of blood flow under these conditions. L-Arginine is the precursor of nitric oxide, an endogenous messenger molecule involved in a variety of endothelium-mediated physiological effects in the vascular system. In the present study, we investigated the effect of oral administration of L-arginine (3 g/day) on the intracellular redox status of the patients of ischemic cardiomyopathy aged 45-60 yrs. The enzymatic and non-enzymatic antioxidant parameters like superoxide dismutase, catalase, total thiols (TSH) and ascorbic acid along with pro-oxidant parameters, such as xanthine oxidase, as well as index of oxidative stress as protein carbonyl content and malondialdehyde (a marker of lipid peroxidation) were investigated in the plasma and RBC lysate. L-Arginine (3 g/day) administration was found to improve the levels of these parameters in the patients and regulate the blood flow, as evident by the improved blood pressure of the patients. Thus, it is inferred that L-arginine attenuates the oxidative stress conditions along with maintaining the blood pressure rate of patients suffering from cardiomyopathy.

Therapeutic role of L-arginine on free radical scavenging system in ischemic heart diseases.

Increased production of free radicals under oxidative stress conditions plays a vital role in the impairment of endothelial function and also in the pathogenesis of ischemic heart diseases. Ischemia, followed by reperfusion, leads to the exacerbated formation of oxy- free radicals. These reactive oxygen species through a chain of reactions damage the cardiomyocytes and cause more injury to the myocardium. L-Arginine is reported to act as free radical scavenger, inhibits the activity of pro-oxidant enzymes and thus acts as an antioxidant and these roles of L-arginine are mediated by nitric oxide (NO). In the present study, the effect of oral administration of L-arginine (3 g/day for 7 days) on some antioxidant enzymes, total thiols, lipid peroxidation measured as malondialdehyde (MDA), and plasma ascorbate levels in myocardial ischemic patients was investigated. We observed an increase in the activity of superoxide dismutase (SOD), total thiols (T-SH) and plasma ascorbate levels and a decrease in the activity of xanthine oxidase (XO), MDA levels, carbonyl content and serum cholesterol in the patients on oral administration of L-arginine. The present study demonstrates that L-arginine administration may be beneficial to patients with myocardial ischemic disorders, such as acute myocardial infarction and acute angina.

Oxidant stress due to non ionic low osmolar contrast medium in rat kidney.

Background & objectives: Contrast media may cause contrast-induced nephropathy (CIN) in risk group. This study was taken up to establish possible effects of non ionic low osmolar contrast medium administration on oxidant/antioxidant status and nitric oxide (NO) levels in rat kidney tissues. Methods: Fourteen female, 14 wk old Wistar-albino rats were divided into 2 groups of 7 rats each (control and contrast groups). Non ionic low osmolar contrast medium was administered iv to the animals in the contrast group. The day after, animals were sacrificed and malondialdehyde (MDA) and NO levels and activities of antioxidant [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT)] and oxidant [xanthine oxidase (XO)] enzymes were measured in kidney tissues. Serum creatinine levels were measured to evaluate kidney functions. Results: Contrast medium administration caused an increase in MDA levels and a decrease in NO levels in kidney tissues. Interpretation & conclusions: The results suggest that non ionic low osmolar contrast medium administration leads to accelerated oxidant reactions and decreased NO level in rat kidney tissues. Further studies need to be done to assess the role of these changes in CIN.

Lentinula edodes (Shiitake) mushroom extract protects against hydrogen peroxide induced cytotoxicty in peripheral blood mononuclear cells.

Lentinula edodes (Berk) Pegler, commonly known as Shiitake mushroom has been used as medicinal food in Asian countries, especially in China and Japan and is believed to possess strong immunomodulatory property. In the present study, the methanolic extract of the fruit bodies of L. edodes was investigated for cytoprotective effect against H2O2-induced cytotoxicity in human peripheral blood mononuclear cells (PBMCs) by measuring the activities of xanthine oxidase (XO) and glutathione peroxidase (GPx) . H2O2 at a concentration of 5 μM caused 50% inhibition of PBMCs viability. The extract improved the PBMC viability and exerted a dose-dependent protection against H2O2-induced cytotoxicity. At 100 μg/ml of extract concentration, the cell viability increased by 60% compared with the PBMCs incubated with H2O2 alone. The extract also inhibited XO activity in PBMC, while showing moderate stimulatory effect on GPx. However, in the presence of H2O2 alone, both the enzyme activities were increased significantly. The GPx activity increased, possibly in response to the increased availability of H2O2 in the cell. When the cells were pretreated with the extract and washed (to remove the extract) prior to the addition of H2O2, the GPx and XO activities as well as the cell viability were comparable to those when incubated with the extract alone. Thus, it is suggested that one of the possible mechanisms via which L. edodes methanolic extract confers protection against H2O2-induced oxidative stress in PBMC is by inhibiting the superoxide-producing XO and increasing GPx activity which could rapidly inactivate H2O2.

Activity of polyphenolic plant extracts as scavengers of free radicals and inhibitors of xanthine oxidase

The free radical scavenging activity of ethanolic and aqueous extracts from six plants has been evaluated, in vitro, using 1, 1-diphenyl-2-picrylhydrazyl [DPPH] radical scavenging method and compared with ascorbic acid, quercetin. kaempferol and apigenin. Extracts of Pistacia integerrima leaves and galls exhibited highest DPPH radical scavenging activity among all the plants extracts examined. Carissa carandas from Apocynaceae and Vitex negundo of Verbenaceae family also demonstrated DPPH radical scavenging activity. Inhibitory effects towards the in-vitro reaction of hypoxanthine and xanthine oxidase [XO] was also carried out in the presence of plant extract, aglycones quercetin, kaempferol and apigenin along with allopurinol The extracts from Pistacia integerrima galls exhibited highest XO inhibitory activity than leaves of Pistacia integerrima and other plant extracts, was comparable with aglycones but less than allopurniol, a pharmacologically different structure. The increased DPPH radical scavenging and XO inhibitors activity by Pistacia integerrima galls may be partly exerted by flavonoids produced during stress of an insect aphids [Pemphogus species] attack on the leaves. The results indicate that Pistacia integerrima may be a good candidate for future studies in vivo models of gout and hyperuricemia

Antioxidant effect of curcumin in selenium induced cataract of Wistar rats.

Wistar rat pups treated with curcumin, a natural constituent of Curcuma longa before being administered with selenium showed no opacities in the lens. The lipid peroxidation, xanthine oxidase enzyme levels in the lenses of curcumin and selenium co-treated animals were significantly less when compared to selenium treated animals. The superoxidase dismutase and catalase enzyme activities of curcumin and selenium co-treated animal lenses showed an enhancement. Curcumin co-treatment seems to prevent oxidative damage and found to delay the development of cataract.

Lupeol, a triterpene, prevents free radical mediated macromolecular damage and alleviates benzoyl peroxide induced biochemical alterations in murine skin.

In our earlier communication we have shown that Lupeol inhibits early responses of tumour induction in murine skin. The free radical mediated damage to the cellular macromolecules such as DNA, proteins, lipids and alteration in the activities of quinone reductase and xanthine oxidase are important biochemical parameters of tumor development. The suppression of free radical mediated damage to cellular macromolecules and induction of quinone reductase along with depletion of xanthine oxidase are prominent characteristics of chemopreventive agents. In the present investigation, we have elucidated the mechanism of action of lupeol (Lup-20 (29)-en-3beta-ol), a triterpene found in moderate amount in many vegetables, fruits and anti-tumor herbs. In the present investigation, lupeol significantly reduced the free radical mediated DNA-sugar damage and microsomal lipid peroxidation in an iron/ascorbate free radical generating system in vitro. Benzoyl peroxide, a known free radical generating tumor promoter mediated oxidation of proteins and modulation in the activities of quinone reductase as well as xanthine oxidase was significantly prevented by lupeol when tested on murine skin in vivo. It was concluded from this study that lupeol acts as an effective chemopreventive agent against cutaneous toxicity.

Lipid peroxidation and antioxidant enzyme levels of intestinal renal and muscle tissues after a 60 minutes exercise in trained mice.

To investigate the effect of blood perfusion difference on oxidant status, mice were trained by a 7-week running program. Two days after the last training session, mice were exercised for 60 minutes at the same training intensity. Changes in the concentration of thiobarbituric acid reactive substance (TBARS), as an index of lipid peroxidation, in intestine, kidney and muscle, were studied in trained mice immediately (0 h), 3 h and 24 h after the running exercise and in unexercised control group. The activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and xanthine oxidase (XO) were determined in these tissues. Tissue SOD activities were unaffected by the exercise. Muscle GPx activity increased after exercise (0 h and 3 h group, P < 0.01) and returned to control levels at 24 h, but there was not any significant difference in intestinal and renal tissues. Renal tissue XO activity could not be determined. There was not any significant difference among groups in intestinal tissue XO activity. The activity of XO was decreased only in skeletal muscle at 0 h (P < 0.05). TBARS levels of exercised groups were higher than control in muscle (P < 0.01). Intestinal TBARS levels decreased at 0 h (P < 0.05), than reached to control level. Renal TBARS levels of 0 h and 24 h group was higher than control (P < 0.01, P < 0.01 respectively). The results show that a long distance running exercise may cause lipid peroxidation damage in skeletal muscle and kidney.

Dissociation of milk xanthine oxidase and effect of some potent inhibitors on its catalytic function

Milk xanthine oxidase [XO] was prepared and purified by gel chromatographic analysis. Chromatographic runs were made on sephadex G200 column saturated with urea. The elution profile recorded two peaks: Dimeric and monomeric xanthine oxidase, respectively. The inhibitory effect of bisulfite, folate and ascorbate on the catalytic activity of xanthine oxidase was monitored by incubating the enzyme with different concentrations of these inhibitors. On assaying aliquot sample of the incubation mixture in a standardized method, a pronounced lag in rate production was recorded spectrophotometrically at 290 nm. XO activity increased from zero until substrate became limiting, but in case of inhibition the rate of catalysis never reached maximal velocity compared to control. Besides the inhibitory mechanism of morin and myricetin as flavonoids on XO activity were also studied by kinetic analysis. It was concluded that bisulfite, folate, ascorbate and flavonoids could be utilized in chemotherapy of hyperuricemia or gout

Immobilization of xanthine oxidase on a polyaniline silicone support

A polyaniline silicone support to immobilize xanthine oxidase is proposed as a reactor coil to monitor the action of xanthine oxidase on hypoxanthine, xanthine and 6-mercaptopurine. A purified xanthine oxidase immobilized on this support lost 80 per cent of the initial activity after 12 min of use. Co-immobilization of superoxide dismutase and catalase increased the stability of immobilized xanthine oxidase so that the derivative maintained 79 per cent of its initial activity after 4.6 h of continuous use in which 1.5 mumol purine bases were converted by the immobilized enzyme system. There is no evidence of either polyaniline or protein leaching from the coil during 3 h of continuous use. When solutions (10 ml) of hypoxanthine, xanthine and 6-mercaptopurine were circulated individually through the xanthine oxidase-superoxide dismutase-catalase-polyaniline coil (1 mm internal diameter and 3 m in length, 3 ml internal volume) activities of 8.12, 11.17 and 1.09 nmol min-1 coil-1, respectively, were obtained. The advantages of the reactor configuration and the redox properties of the polymer, particularly with respect to immobilized oxidoreductases, make this methodology attractive for similar enzyme systems. This immobilized enzyme system using polyaniline-silicone as support converted 6-mercaptopurine to 6-thiouric acid with equal efficiency as resins based on polyacrylamide and polyamide 11.

Action of immobilized xanthine oxidase on purines

Xanthine oxidase was covalently immbolized on polyacrylamide gel beads, polyamide- 11 and dacron. Hypoxanthine (15 ml of 200 µM), prepared in 0.1 M phosphate buffer, pH 8.0, was circulated through a column containing 1.0g derivatized enzyme at a flow rate of 1.0 ml/min at 28§C. Specific activities of 0.660, 0.072 and 0.016 Units/mg of protein were demonstrable for the polyacrylamide gel beads, dacron and polyamide-11 derivatives, respectively. The action of these water insoluble enzyme derivatives on 6 mercaptopurine (15 ml of 660 µM) was also investigated, under the same experimental conditions, showing specific activites of 0.063 Units/mg, 0.574 µUnits/mg and 0.118 µUnitis/mg, respectively. The 6-mercaptopurine oxidative pathway catalyzed by immobilized xanthine oxidase on dacron stopped at the intermediate compound 6-mercaptopurine oxidative on dracon stopped at the intermediate compound, 6-mercapto-8-hydroxypurine, so that no 6-thiouric acid was produced, whereas the immobilized preparations using polyacrylamide gel beads and polyamide-11 behaved like the soluble enzyme, namely, 6-thiouric acid was the final product. The behavior of dracon-xanthine oxidase immobilized on these three supports was similar to the soluble enzyme. However, although its oxidation is stoichiometric for polyacrylamide gel beads and polyamide- 11 derivatives, and no xanthine formation is observed (steady-state equilibrium), under the action of the enzymedacron derivative the xanthine formation rate (0.164 µUnits/mg) is higher than the uric acid formation rate (0.017 µUnits/mg) compared to the hypoxanthine consumption (0.072 µUnits/mg). These findings suggest again that xanthine oxidase-dacron derivative is limited to the catalysis of oxidation of hypoxanthine carbon atom number 2 as in 6-mercaptopurine

Interrelation of xanthine oxidase and dehydrogenase and L-gulonolactone oxidase in animal tissues.

There is a correlation between phylogeny and the activities of L-gulonolactone oxidase (LGO), the key enzyme responsible for ascorbic acid (AH2) synthesis in animals and total xanthine oxidase and dehydrogenase [XOD(D/O)], the enzyme responsible for the production of endogenous superoxide radical (O2-.). LGO appears in the kidneys of amphibians and reptiles but livers of mammals. XOD(D/O) also is present mainly in the kidneys of amphibians and reptiles and livers of mammals. AH2 is a potential scavenger of O2-. and it appears that tissue specific expression of LGO takes place to counteract the endogenous O2-. toxicity. The interrelation of XOD(D/O) and LGO was also observed in the liver of rats during prenatal to postnatal development.

Reversal of changes of lipid peroxide, xanthine oxidase and superoxide dismutase by cardio-protective drugs in isoproterenol induced myocardial necrosis in rats.

In myocardial necrosis produced by isoproterenol (beta-adrenergic agonist) marked increase in creatine phosphokinase, phospholipase and significant decrease in cardiac glycogen and phospholipid levels were observed. The enhanced levels of lipid peroxides, xanthine oxidase activity and lowering of superoxide dismutase may lead to excessive formation of free radicals resulting in cardiac cell damage. Nifedipine--a calcium antagonist, Propranolol--a beta-blocker and guggulsterone a lipid lowering agent showed marked reversal of these metabolic changes related to ischemia induced by isoproterenol.

Actividad de la xantina oxidasa en productos lácteos de venta en Panamá / Activity of the xanthine oxidase in dairy products of sale in Panama

Se determinó polarográficamente la actividad de la Xantina Oxidasa (XO) en 67 productos lácteos de venta en Panamá. Las leches líquidas tenían entre 0.31 y 26.10 ImU/ml. Las leches en polvo tenían entre 1.41 y 19.50 ImU/ml. Los yorgurts y la leche de cabra pasteurizada tenían actividades menores de 1.30 ImU/ml. Las margarinas, los helados y las leches evaporadas casi eran exentas de la actividad de XO. Entre los quesos, los blancos nacionales fueron los que mostraron la más alta actividad de XO, con un promedio de 346.28 ImU/ml, entre 253.14 y 498.54 ImU/ml