OBJECTIVETo evaluate the inhibitory effect of Gnaphalium affine extracts on xanthine oxidase (XO) activity in vitro and to analyze the mechanism of this effect.

METHODSIn this in vitro study, Kinetic measurements were performed in 4 different inhibitor concentrations and 5 different xanthine concentrations (60, 100, 200, 300, 400 Μmol/L). Dixon and Lineweaver-Burk plot analysis were used to determine Ki values and the inhibition mode for the compounds isolated from Gnaphalium affine extract.

RESULTSFour potent xanthine oxidase inhibitors were found in 95% ethanolic (v/v) Gnaphalium affine extract. Among them, the flavone Eupatilin exhibited the strongest inhibitory effect on XO with a inhibition constant (Ki) of 0.37 Μmol/L, lower than the Ki of allopurinol (4.56 mol/L), a known synthetic XO inhibitor. Apigenin (Ki of 0.56 Μmol/L, a proportion of 0.0053‰ in Gnaphalium affine), luteolin (Ki of 2.63 Μmol/L, 0.0032‰ in Gnaphalium affine) and 5-hydroxy-6,7,3',4'-tetramethoxyflavone (Ki of 3.15 Μmol/L, 0.0043‰ in Gnaphalium affine) also contributed to the inhibitory effect of Gnaphalium affine extract on XO activity.

CONCLUSIONSThese results suggest that the use of Gnaphalium affine in the treatment of gout could be attributed to its inhibitory effect on XO. This study provides a rational basis for the traditional use of Gnaphalium affine against gout.

Flavonoids ; pharmacology ; Gnaphalium ; chemistry ; Xanthine Oxidase ; antagonists & inhibitors

A double targets of high throughput screening model for xanthine oxidase inhibitors and superoxide anion scavengers was established. In the reaction system of xanthine oxidase, WST-1 works as the probe for the ultra oxygen anion generation, and product uric acid works as xanthine oxidase activity indicator. By using SpectraMax M5 continuous spectrum enzyme sign reflectoscope reflector, the changes of these indicators' concentration were observed and the influence factors of this reaction system to establish the high throughput screening model were studied. And the model is confirmed by positive drugs. In the reaction system, the final volume of reaction system is 50 μL and the concentrations of xanthine oxidase is 4 mU x mL(-1), xanthine 250 μmol x L(-1) and WST-1 100 μmol x L(-1), separately. The Z'-factor of model for xanthine oxidase inhibitors is 0.537 4, S/N is 47.519 9; the Z'-factor of model for superoxide anion scavengers is 0.507 4, S/N is 5.388 9. This model for xanthine oxidase inhibitors and superoxide anion scavengers has more common characteristics of the good stability, the fewer reagent types and quantity, the good repeatability, and so on. And it can be widely applied in high-throughput screening research.
Enzyme Inhibitors ; pharmacology ; Free Radical Scavengers ; pharmacology ; High-Throughput Screening Assays ; Superoxides ; Uric Acid ; Xanthine ; Xanthine Oxidase ; antagonists & inhibitors

Eight compounds,(R)-2-[5-(methoxycarbonyl)-4-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl]acetic acid(1),(3S,4R)-3,4-dihydro-3,4-epoxy-5-hydroxynaphthalen-1(2H)-one(2),(-)-mitorubrinol(3),(-)-mitorubrin(4),(±)-asperlone A(5), terreusinone(6), verrucisidinol(7) and cerebroside C(8) were isolated from the endophytic fungus Talaromyces purpurogenus by using various column chromatographic techniques. Their structures were identified by NMR, MS, CD and optical rotation. Compounds 1 and 2 were new compounds. Their anti-diabetic activities in vitro were evaluated, and compound 1 showed moderate inhibitory activity toward XOD at 10 μmol·L~(-1) with the inhibition rate of 69.9%.
Endophytes/chemistry* ; Hypoglycemic Agents/chemistry* ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Secondary Metabolism ; Talaromyces/chemistry* ; Tylophora/microbiology* ; Xanthine Oxidase/antagonists & inhibitors*

To determine whether oxygen free radicals are responsible for the pathogenesis of the cholestasis induced by ligation of common bile duct (CBD) variables which reflect the hepatic function in the serum, the amount of superoxide radical production, and xanthine oxidase(XO) activity were studied. The activity of serum alanine aminotransferase, bilirubin level in the serum and the amount of superoxide radical production were lower in a CBD ligation with allopurinol treated group than in a CBD ligation without allopurinol treated group. Abnormalities of the microscopic structures were reduced in a CBD ligation with allopurinol treated group than in a CBD ligation without allopurinol treated group. Allopurinol, an inhibitor of XO, prevented the hepatic damage induced by CBD ligation through the inhibition of XO. These experiments demonstrate that oxygen free radicals are responsible for the pathogenesis of the cholestatic liver.
Allopurinol/*pharmacology ; Animal ; Bile Ducts ; Cholestasis/*pathology ; Enzyme Inhibitors/*pharmacology ; Free Radicals ; Ligation ; Liver/*pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Superoxides/metabolism ; Xanthine Oxidase/analysis/*antagonists & inhibitors

Animals ; Chronic Disease ; Febuxostat ; Heart Failure ; drug therapy ; metabolism ; Humans ; Reactive Oxygen Species ; Thiazoles ; pharmacology ; therapeutic use ; Xanthine Oxidase ; antagonists & inhibitors

Human xanthine oxidase is considered to be a target for therapy of hyperuricemia. Cichorium intybus is a Chinese plant medicine which widely used in Xinjiang against various diseases. In order to screen the inhibitors of xanthine oxidase from C. intybus and to explore main pharmacological actions of cichory a compound collection of C. intybus was built via consulting related references about chemical research on cichory. The three-dimensional crystal structure of xanthine oxidase (PDB code: 1N5X) from Protein Data Bank was downloaded.. Autodock 4.2 was employed to screen the inhibitors of xanthine oxidase from cichory 70 compounds were found to possess quite low binding free energy comparing with TEI (febuxostat). C. intybus contains constituents possessing potential inhibitive activity against xanthine oxidase. It can explain the main pharmacological actions of cichory which can significantly lower the level of serum uric acid.
Chicory ; chemistry ; Databases, Protein ; Drugs, Chinese Herbal ; chemistry ; Enzyme Inhibitors ; chemistry ; Humans ; Molecular Docking Simulation ; Molecular Structure ; Xanthine Oxidase ; antagonists & inhibitors ; metabolism

This paper is aimed to study the metabolic kinetics of nicousamide in rat liver microsomes and cytosol and to identify the major metabolite and drug metabolizing enzymes involved in the metabolism of nicousamide in rat and human liver microsomes by selective inhibitors in vitro. The concentration of nicousamide was determined by HPLC-UV method. The metabolite of nicousamide in rat and human liver microsomes was isolated and identified by LC-MS/MS. The major metabolite of nicousamide in rat and human liver microsomes was identified to be 3-(3'-carboxy-4'-hydroxy-anilino-carbo-)-6-amino-7-hydroxy-8-methyl-coumarin (M1). The metabolite of nicousamide in rat plasma, urine, bile and liver was consistent with M1. The metabolism of nicousamide can be catalyzed by several reductases, including CYP450 reductases, cytochrome b5 reductases and CYP2C6 in rat liver microsomes, as well as xanthine oxidase and DT-diaphorase in rat liver cytosol.
Adenosine Monophosphate ; pharmacology ; Allopurinol ; pharmacology ; Aniline Compounds ; metabolism ; Animals ; Cimetidine ; pharmacology ; Coumarins ; metabolism ; Cytochrome P-450 Enzyme Inhibitors ; Cytochrome P450 Family 2 ; Cytochrome-B(5) Reductase ; antagonists & inhibitors ; Cytosol ; metabolism ; Dicumarol ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Female ; Humans ; Liver ; cytology ; metabolism ; Male ; Microsomes, Liver ; metabolism ; Mitochondria, Liver ; metabolism ; NAD(P)H Dehydrogenase (Quinone) ; antagonists & inhibitors ; Propylthiouracil ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Steroid 21-Hydroxylase ; antagonists & inhibitors ; Xanthine Oxidase ; antagonists & inhibitors

OBJECTIVETo further investigate the neuroprotective effects of five isoflavonoids from Astragalus mongholicus on xanthine (XA)/ xanthine oxidase (XO)-induced injury to PC12 cells.

METHODSPC12 cells were damaged by XA/XO. The activities of antioxidant enzymes, MTT, LDH, and GSH assays were used to evaluate the protection of these five isoflavonoids. Contents of Bcl-2 family proteins were determined with flow cytometry.

RESULTSAmong the five isoflavonoids including formononetin, ononin, 9, 10-dimethoxypterocarpan-3-O-beta-D-glucoside, calycosin and calycosin-7-O-glucoside, calycosin and calycosin-7-O-glucoside were found to inhibit XA/ XO-induced injury to PC12 cells. Their EC50 values of formononetin and calycosin were 0.05 microg/mL. Moreover, treatment with these three isoflavonoids prevented a decrease in the activities of antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), while formononetin and calycosin could prevent a significant deletion of GSH. In addition, only calycosin and calycosin-7-O-glucoside were shown to inhibit XO activity in cell-free system, with an approximate IC50 value of 10 microg/mL and 50 microg/mL. Formononetin and calycosin had no significant influence on Bcl-2 or Bax protein contents.

CONCLUSIONNeuroprotection of formononetin, calycosin and calycosin-7-O-glucoside may be mediated by increasing endogenous antioxidants, rather by inhibiting XO activities or by scavenging free radicals.

Animals ; Astragalus Plant ; chemistry ; Glucosides ; chemistry ; isolation & purification ; pharmacology ; Glutathione ; metabolism ; Glutathione Peroxidase ; metabolism ; Isoflavones ; chemistry ; isolation & purification ; pharmacology ; PC12 Cells ; Protective Agents ; isolation & purification ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Superoxide Dismutase ; metabolism ; Xanthine Oxidase ; antagonists & inhibitors ; pharmacology

Gnaphalium affine D. Don, a medicinal and edible plant, has been used to treat gout in traditional Chinese medicine and popularly consumed in China for a long time. A detailed phytochemical investigation on the aerial part of G. affine led to the isolation of two new esters of caffeoylquinic acid named (-) ethyl 1, 4-di-O-caffeoylquinate (1) and (-) methyl 1, 4-di-O-caffeoylquinate (2), together with 35 known compounds (3-37). Their structures were elucidated by spectroscopic data and first-order multiplet analysis. All the isolated compounds were tested for their xanthine oxidase inhibitory activity with an in vitro enzyme inhibitory screening assay. Among the tested compounds, 1 (IC 11.94 μmol·L) and 2 (IC 15.04 μmol·L) showed a good inhibitory activity. The current results supported the medical use of the plant.
Adenine ; analogs & derivatives ; chemistry ; isolation & purification ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Enzyme Activation ; drug effects ; Flavonoids ; chemistry ; isolation & purification ; Gnaphalium ; chemistry ; Gout Suppressants ; chemistry ; isolation & purification ; pharmacology ; Hydroxybenzoates ; chemistry ; isolation & purification ; Molecular Structure ; Nuclear Magnetic Resonance, Biomolecular ; Phytochemicals ; chemistry ; isolation & purification ; pharmacology ; Plant Components, Aerial ; chemistry ; Plant Extracts ; chemistry ; isolation & purification ; pharmacology ; Quinic Acid ; analogs & derivatives ; chemistry ; isolation & purification ; Xanthine Oxidase ; antagonists & inhibitors