A hyperuricemic rat model induced by adenine and ethambutol was established to investigate the anti-hyperuricemia activity and its mechanism of the flavonoid extract from saffron floral bio-residues. Sixty-seven SD rats were randomly divided into control group, model group, positive control group, and flavonoid extract groups(with 3 doses), respectively, and each group contained 11 or 12 rats. The hyperuricemic model was established by continuous oral administration of adenine(100 mg·kg~(-1)) and ethambutol(250 mg·kg~(-1)) for 7 days. At the same time, the positive control group was given allopurinol(20 mg·kg~(-1) per day) and the flavonoid extract groups were given the flavonoid extract at doses of 340, 170 and 85 mg·kg~(-1) per day, respectively. On day 8, rat serum, liver, kidney, and intestinal tissues were collected, and the levels of uric acid in serum and tissue, the xanthine oxidase activities and antioxi-dant activities in serum and liver were evaluated, and the kidney histopathology was explored. In addition, an untargeted serum metabolomics study was performed. According to the results, the flavonoid extract effectively reduced the uric acid levels in serum, kidney and ileum and inhibited the xanthine oxidase activities and elevated the antioxidant activities of serum and liver in hyperuricemic rat. At the same time, it reduced the levels of inflammation factors in kidney and protected renal function. Moreover, 68 differential metabolites of hyperuricemic rats were screened and most of which were lipids and amino acids. The flavonoid extract significantly retrieved the levels of differential metabolites in hyperuricemic rats, such as SM(d18:1/20:0), PC[18:0/18:2(92,12Z)], palmitic acid and citrulline, possibly through the following three pathways, i.e., arginine biosynthesis, glycine, serine and threonine metabolism, and histidine metabolism. To sum up, the flavonoid extract of saffron floral bio-residues lowered the uric acid level, increased the antioxidant activity, and alleviated inflammatory symptoms of hyperuricemic rats, which may be related to its inhibition of xanthine oxidase activity and regulation of serum lipids and amino acids metabolism.
Rats ; Animals ; Flavonoids/pharmacology* ; Uric Acid ; Crocus ; Xanthine Oxidase ; Ethambutol/adverse effects* ; Rats, Sprague-Dawley ; Hyperuricemia/drug therapy* ; Kidney ; Antioxidants/pharmacology* ; Plant Extracts/adverse effects* ; Amino Acids ; Adenine/adverse effects* ; Lipids

Background: One of the causes of inflammatory arthritis is excessive production of uric acid or hyperuricemia. It is a painful disease that is treated with a commercial xanthine oxidase inhibitor to decrease uric acid synthesis. However, the treatment is associated with adverse side effects and thus, there is interest in medicinal plants that could have similar therapeutic effects with minimal side effects. There are many reported indigenous plants and trees in the Philippines that are reported to have therapeutic and bioactive compounds. One such plant is Canarium ovatum or locally called pili. This study aimed to determine the antihyperuricemic activity of the ethanolic extract of the leaves of C. ovatum. Objective: Determine the antihyperuricemic activity of the crude ethanolic extract of C. ovatum leaves and its partially purified fractions through inhibition of xanthine oxidase and its effect on the blood uric acid level of oxonate-induced hyperuricemic mice. Methodology: The crude ethanol extract from C. ovatum leaves and its partially purified fractions obtained through column chromatography were tested for their in vitro xanthine oxidase (XO) inhibitory activity by measuring spectrophotometrically the uric acid formation from xanthine as the substrate. The crude ethanol extract and the fraction with the most XO inhibitory activity were then tested for their in vivo XO inhibitory activity in oxonate-induced hyperuricemic mice by measuring their blood uric acid levels using uric acid test strips. Results: The crude ethanolic extract of C. ovatum leaves at 100ppm showed 83.62±2.05% in vitro inhibition of XO while the most active fraction showed 80.30±4.00% inhibition. Both were comparable (p>0.05) to the positive control, allopurinol, which showed 91.47±5.64% inhibition. In vivo, the crude extract and the fraction that showed the highest XO inhibitory activity at 200 mg/kg significantly (p<0.01 and p<0.05) respectively reduced the serum uric acid levels of the hyperuricemic mice one hour after induction as compared to the negative control. Moreover, their antihyperuricemic activity were not statistically significant as compared to that of allopurinol (p<0.0001). Conclusion The crude ethanolic extract of C. ovatum leaves and its most active fraction showed statistically significant in vitro xanthine oxidase inhibition and in vivo antihyperuricemic activity. The activities shown by both crude and active fraction were not statistically different from that determined for allopurinol. Therefore, further studies can be conducted to isolate the most active compound and study its pharmacokinetic properties.
Xanthine Oxidase ; Uric Acid ; Allopurinol

The present study investigated the mechanism of total flavonoids from Ampelopsis grossedentata(AGTF) against gouty arthritis(GA) by network pharmacology and experimental validation. The main active ingredients and targets of AGTF, as well as disease targets, were screened out using relevant databases and literature data. The "protein-protein interaction"(PPI) network and "drug-ingredient-target-pathway" network were constructed, and the potential targets and mechanism of AGTF against GA were predicted. The hyperuricemia(HUA) combined with GA model was induced in rats. The gait behaviors of rats were scored, and ankle swelling degree was observed. The uric acid(UA) level and xanthine oxidase(XOD) activity in the rat serum were detected, and the levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) were measured. The protein expression of toll-like receptor 4(TLR4), myeloid differentiation factor 88(MyD88), and nuclear factor-kappa B(NF-κB) in the synovial tissues of the rat ankle joint was determined by immunohistochemistry. Ten active ingredients of AGTF and 73 candidate targets of AGTF against GA were screened out by network pharmacology. Eighty-six signaling pathways were enriched, including TNF signaling pathway, NF-κB signaling pathway, TLR signaling pathway, Nod-like receptor signaling pathway, and purine metabolism signaling pathway, which were closely related to AGTF against GA. Animal experimental results showed that AGTF could effectively improve the abnormal gait behaviors of GA rats, relieve ankle inflammation, and reduce ankle joint swelling. In addition, AGTF could significantly reduce UA level, inhibit XOD activity, decrease TNF-α, IL-6, and IL-1β content, and down-regulate the expression of TLR4, MyD88, and NF-κB in ankle synovial tissues(P<0.05, P<0.01). The results of network pharmacology and experimental validation are consistent, indicating that AGTF exerts its therapeutic effect on GA by regulating UA metabolism, improving abnormal UA level, reducing the release of inflammatory factors, and regulating immunity and the TLR4/MyD88/NF-κB inflammatory pathway.
Ampelopsis/chemistry* ; Animals ; Arthritis, Gouty/drug therapy* ; Flavonoids/therapeutic use* ; Interleukin-1beta/metabolism* ; Interleukin-6/metabolism* ; Myeloid Differentiation Factor 88/metabolism* ; NF-kappa B/metabolism* ; NLR Proteins/metabolism* ; Rats ; Toll-Like Receptor 4/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Uric Acid ; Xanthine Oxidase

The present study analyzed and identified the chemical constituents from ethyl acetate(EA) extract of Taxilli Herba with UPLC-Q-Exactive-MS and screened active xanthine oxidase(XO) inhibitors with HPLC. The analysis was performed on an Hypersil GOLD C_(18) reversed-phase column(2.1 mm×50 mm, 1.9 μm), with the mobile phase of water containing 1% formic acid(A) and methanol(B) under gradient elution, the flow rate of 0.3 mL·min~(-1), and the injection volume of 5 μL. ESI source was used for MS and the compounds were collected in positive and negative ion modes. Xcalibur 4.1 was used to analyze the retention time, accurate relative molecular weight, and fragmentation of the compounds. The inhibitory activity of some known compounds on XO was screened by HPLC. Thirty chemical constituents were identified, including phenolic acids and flavonoids by experimental data combined with information of standards, data reported previously, and databases, such as MzCloud and ChemSpider. The activities of 10 chemical components were screened. Gallic acid and naringenin chalcone had strong inhibitory activities on XO with IC_(50) of 57 μg·mL~(-1) and 108 μg·mL~(-1). UPLC-Q-Exactive-MS allows the accurate, rapid, and comprehensive identification of main chemical constituents from Taxilli Herba. Gallic acid and naringenin chalcone may be the active components of XO inhibitors.
Acetates ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal/chemistry* ; Tandem Mass Spectrometry ; Xanthine Oxidase

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.
Chromatography, Liquid ; Chrysanthemum ; Flavonoids ; Kinetics ; Pharmaceutical Preparations ; Tandem Mass Spectrometry ; Xanthine Oxidase/metabolism*

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*

In the present study, two new acetylene conjugate compounds, dibutyl (2Z, 6Z)-octa-2, 6-dien-4-yne dioate (1), and dibutyl (2E, 6E)- octa-2, 6-dien-4-yne dioate (2), were isolated from the dry stem leaves of Viscum album, along with nine known compounds (3 - 11). Their structures were confirmed on the basis of spectroscopic data. Compounds 1 and 8 showed antioxidant activity against xanthine oxidase (XOD) and 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydroxyl (DPPH), with the IC of 1.22 and 1.33 μmol·L, and the SC of 4.34 and 8.22 μmol·L, respectively.
Acetylene ; chemistry ; Antioxidants ; chemistry ; pharmacology ; Biphenyl Compounds ; chemistry ; Molecular Structure ; Picrates ; chemistry ; Plant Extracts ; chemistry ; pharmacology ; Plant Leaves ; chemistry ; Viscum album ; chemistry ; Xanthine Oxidase ; chemistry

BACKGROUND: Xanthine derivatives have been used to treat a variety of medical conditions including respiratory disease and neural degeneration. However, few studies have reported their effects on bone regeneration. Therefore, we investigated the effects of KPR-A148, a synthetic xanthine derivative on osteoblast differentiation in vitro and bone regeneration in vivo. METHODS: The cytotoxicity of KPR-A148 was evaluated using MC3T3-E1 cells by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltertrazolium bromide assay. The effects of KPR-A148 on osteoblast differentiation were examined by alkaline phosphatase staining, Alizarin red S staining, and real-time PCR of osteoblast differentiation marker genes. To investigate the effects of KPR-A148 on in vivo bone regeneration, a KPR-A148-containing collagen sponge was implanted into a mouse calvarial defect and KPR-A148 was injected twice, weekly. Bone regeneration was evaluated quantitatively by micro-CT and qualitatively by hematoxylin and eosin, as well as Masson's Trichrome staining. RESULTS: KPR-A148 did not show toxicity in the MC3T3-E1 cells and promoted osteoblast differentiation in a concentration-dependent manner. 10 µM of KPR-A148 showed the most significant effect on alkaline phospatase staining and matrix mineralization. KPR-A148 increased the expression of osteoblast marker genes in both the early and late stages of differentiation. In addition, KPR-A148 significantly induced new bone formation in the calvarial defect model. CONCLUSION: These results demonstrate that KPR-A148 strongly induces osteoblast differentiation and new bone formation. Therefore, it could be used as a potential therapeutic agent for regenerating bone following its destruction by disease or trauma.
Alkaline Phosphatase ; Animals ; Bone Regeneration ; Collagen ; Eosine Yellowish-(YS) ; Hematoxylin ; In Vitro Techniques ; Mice ; Miners ; Osteoblasts ; Osteogenesis ; Porifera ; Real-Time Polymerase Chain Reaction ; Xanthine

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

The prevalence of gout is increasing worldwide, and control of serum uric acid level has been regarded as one of the therapeutic methods for gout. Inhibition of xanthine oxidase (XO) activity which can oxidize hypoxanthine to uric acid has been commonly proposed to decrease serum uric acid level. The aim of this study was to demonstrate the hypouricemic effect of ethanol extract of Aster glehni leaves (EAG) by in vitro and in vivo study in potassium oxonate (PO)-induced hyperuricemic rats. EAG possessed 132.5 ± 6.8 mg QE/g of total flavonoid and showed antioxidant activity. EAG showed in vitro and in vivo inhibitory activity against XO and significantly decreased serum uric acid level in PO-induced hyperuricemic rats without liver toxicity. These results show that EAG significantly attenuates hyperuricemia by inhibiting XO activity, which resulted in the decrease of serum uric acid level. Therefore, EAG might possess a potential therapeutic ability for improving gout.
Animals ; Ethanol* ; Gout ; Hyperuricemia ; Hypoxanthine ; In Vitro Techniques ; Liver ; Potassium* ; Prevalence ; Rats* ; Uric Acid ; Xanthine Oxidase