Antioxidant activity and in vitro and in silico gout inhibitory effect of benzylideneacetophenone derivatives

A method based on enzyme blocking combined with ultrafiltration liquid chromatography-mass spectrometry (LC-MS) has been developed to identify xanthine oxidase (XOD) inhibitors in the roots of Lindera reflexa Hemsl (LR) and determine their binding positions. Allopurinol and febuxostat, known XOD inhibitors, which occupy different binding positions in XOD, were used as blockers and pre-incubated with XOD. Then the LR extract was incubated without XOD, and with XOD, allopurinol-blocked XOD and febuxostat-blocked XOD before ultrafiltration LC-MS was performed. By comparing the chromatographic profiles of the incubation samples, not only the ligands, but also the binding position of these ligands with XOD could be determined. Finally, three compounds, pinosylvin, pinocembrin and methoxy-5-hydroxy-trans-stilbene, were identified as potential XOD inhibitors and the binding modes of these three compounds were shown to be similar to those of febuxostat. To verify the XOD inhibitory activity of the screened compounds, the microplate method and molecular docking in silico were used to evaluate the enzyme inhibitory activities and the binding positions with XOD. The results showed that the developed method could screen for XOD ligands in LR extracts and also determine the binding positions of the ligands. To our knowledge, this is the first report of the XOD inhibitory activity of these three compounds.

Development and optimization of Fe₃O₄@SiO₂-xanthine oxidase magnetic nanoparticles for rapid screening of XOD inhibitors from Trichosanthes anguina (snake gourd) with molecular docking approach.

The present study was designed to evaluate the xanthine oxidase (XO) inhibitory and antioxidant activities of 30 bioactive compounds present in edible food plants for the possible treatment of hyperuricemia. The XO inhibitory, SO and DPPH radical scavenging activities of selected dietary polyphenols were determined by using colorimetric assays. The molecular docking analysis was performed to evaluate the insight into inhibitory mode of action of bioactive compounds against XO. The results show that apigenin, galangin, kaempferol, quercetin, genistein and resveratrol potently inhibit XO enzyme among all tested compounds. Flavonoids exhibit higher, anthocyanins and hydroxycinnamic acids moderate, maslinic acid, ellagic acid, salicylic acid, [6]-gingerol and flavan-3-ols showed weak XO inhibitory activity. The results of molecular docking study revealed that these bioactive compounds bind with the active site of XO and occupy the active site which further prevents the entrance of substrate and results in the inhibition of XO. Inhibition of XO gives a robust biochemical basis for management of hyperuricemia, gout and other associated diseases via controlling uric acid synthesis.

In the present study, in silico predictions and molecular docking were performed on five clerodane diterpenes (1–5) from Polyalthia longifolia seeds to evaluate their potential as xanthine oxidase (XO) inhibitors. The initial screening was conducted by target prediction using TargetNet web server application and only compounds 3 and 4 showed a potential interaction with XO. Compounds 3 and 4 were subsequently subjected to in silico analyses on XO protein structure (PDB: 1N5X) using Schrödinger Release 2020–3 followed by structural modeling & molecular simulation studies to confirm the initial prediction result and identify the binding mode of these compounds to the XO. Molecular docking results revealed that compounds 3 (-37.3 kcal/mol) and 4 (-32.0 kcal/mol) binds more stably to XO than the reference drug allopurinol (-27.0 kcal/mol). Interestingly, two residues Glu 802 and Thr 1010 were observed as the two main H-bond binding sites for both tested compounds and the allopurinol. The center scaffold of allopurinol was positioned by some π-π stacking with Phe 914 and Phe 1009, while that of compounds 3 and 4 were supported by many hydrophobic interactions mainly with Leu 648, Phe 649, Phe 1013, and Leu 1014. Additionally, the docking simulation predicted that the inhibitory effect of compounds 3 and 4 was mediated by creating H-bond with particularly Glu 802, which is a key amino acid for XO enzyme inhibition. Altogether, in vitro studies showed that compounds 3 and 4 had better inhibitory capacity against XO enzyme with IC50 values significantly (p < 0.001) lower than that of allopurinol. In short, the present study identified cleroda-4(18),13-dien-15,16-olide as novel potential XO inhibitors, which can be potentially used for the treatment of gout.

Opuntia ficus-indica fruit (OFI) is rich in bioactive compounds, which can promote human health. In this work, the purified OFI extract was prepared from OFI and its bioactivities were investigated. Xanthine oxidase (XOD) and α-glucosidase (α-Glu) inhibitors of the purified OFI extract were screened and identified by bio-affinity ultrafiltration combined with UPLC-QTRAP-MS/MS technology. The inhibitory effect of these inhibitors on enzymes were verified, and the potential mechanism of action and binding sites of inhibitors with enzymes were revealed based on molecular docking. The results showed that the total phenolic content of the purified OFI extract was 355.03mg GAE/g DW, which had excellent antioxidant activity. Additionally, the extract had a certain inhibitory effect on XOD (IC50=199.00±0.14µg/mL) and α-Glu (IC50=159.67±0.01µg/mL). Seven XOD inhibitors and eight α-Glu inhibitors were identified. Furthermore, XOD and α-Glu inhibition experiments in vitro confirmed that inhibitors such as chlorogenic acid, taxifolin, and naringenin had significant inhibitory effects on XOD and α-Glu. The molecular docking results indicated that inhibitors could bind to the corresponding enzymes and had strong binding force. These findings demonstratethat OFI contains potential substances for the treatment of hyperuricemia and hyperglycemia.

In vivo anti-hyperuricemic and xanthine oxidase inhibitory properties of tuna protein hydrolysates and its isolated fractions

Metabolomic profiling of Piper sarmentosum Roxb. Extracts reveals potent xanthine oxidase inhibition and anti-inflammatory effects.

Context: Hyperuricemia is a metabolic syndrome characterized by a high serum uric acid level with an increased risk of gout, diabetes, cardiovascular and renal disease. Allopurinol is a widely known xanthine oxidase inhibitor that lowers serum uric acid production. Documented adverse effects of the drug raise the need for a natural alternative for xanthine oxidase inhibitor that is safe and effective. This research is driven to address the need by capitalizing on previous studies made on the Dillenia species showing nephroprotective and antihyperuricemic activity. Aims: To evaluate the antioxidant capacity, xanthine oxidase inhibitory activity, and acute toxicity of Dillenia philippinensis; a plant locally known as Katmon and endemic to the Philippines. Methods: The D. philippinensis ethanolic leaf extract (DPELE) was partitioned using solvents of different polarities, hexane, ethyl acetate, and butanol. The antioxidant activity of the DPELE and the sub-extracts was assessed using nitric oxide, hydrogen peroxide, and hydroxyl radical scavenging assays against ascorbic acid, while the xanthine oxidase inhibitory activity was examined against allopurinol as a standard. The acute toxicity was performed using Sprague-Dawley rats following OECD 425 guidelines. Results: The D. philippinensis ethyl acetate fraction (DPEAF) had the highest scavenging activity of nitric oxide, hydrogen peroxide, and hydroxyl (IC50 = 210.00, 70.92 and 59.88 µg/mL) free radicals, respectively. Also, the xanthine oxidase dose-dependent inhibitory activity was observed to be highest in the DPEAF with an IC50 = 23.09 µg/mL. Lastly, neither toxicity nor mortalities were observed in rats treated with the DPELE and the DPEAF for 14 days. The approximate lethal dose for DPELE and DPEAF was higher than 2000 mg/kg BW. Conclusions: This study shows that the DPELE and DPEAF are potential sources of natural antioxidants and xanthine oxidase inhibitors, which may be used in the treatment of hyperuricemia and are presumed safe to be used orally.

This thesis contains the results of a QSAR analysis of the interaction of bovine milk xanthine oxidase with two azaheterocyclic compounds, namely the 6-arylpteridin- 4-ones and the 8-arylhypoxanthines. Xanthine oxidase has active sites for various substrates. The studies done for this thesis were of the active site connected to the molybdenum cofactor.Chapter 2 contains a description of how the 6-arylpteridin-4-ones and the 8-arylhypoxanthines were prepared. To synthesize the congeneric series of pteridines, the Gabriel-Isay method was used. This method, in which 4,5-diamino-6-hydroxypyrimidin reacts with an arylglyoxal, invariably led to contamination of the reaction product with isomeric 7-arylpteridin-4-ones. Multiple recrystallization from a DMSO-water solution minimizes contamination of the product to less than 5%. To synthesize the 8-arylhypoxanthines, benzoic acid derivatives were used instead of arylglyoxal derivatives.The 6-arylpteridin-4-ones are good inhibitors of both free and immobilized xanthine oxidase (Chapter 3). To study the inhibitory properties of these compounds as expressed by their K i values, a QSAR equation was calculated. The equation shows that the electronic character of the 6-arylpteridin-4-one substituents does not influence the inhibition of xanthine oxidase; it is governed only by steric factors. The QSAR equation also shows that the effectiveness of an inhibitor is reduced by large spherical substituents like t -butyl and enhanced by rod-shaped substituents like n -butyl. For immobilized xanthine oxidase, an additional factor, namely the hydrophobic parameter π, is essential for the equation. The explanation for this lies in the hydrophilic character of the Sepharose matrix that is used to immobilize the enzyme.The 8-arylhypoxanthines were synthesized and tested for their effectiveness as substrates of xanthine oxidase (Chapter 4). During synthesis, the 8-arylhypoxanthines are converted into the corresponding 8-arylxanthines. To characterize the product, the 8-phenylhypoxanthine was incubated with immobilized xanthine oxidase. The product of the enzymatic reaction was then isolated and characterized as 8- phenylxanthine by 13C-NMR and IR spectroscopy. The oxidation of these compounds is so slow that a detailed study was done only for the upsubstituted compound, the p -methyl, and the p -bromo substituted 8-arylliypoxanthines. At a high pH level, V max is considerably higher than at a low level and K m remains essentially the same. V max and K m are both higher at high temperatures.Chapter 5 contains the results of experiments on the inhibition of free xanthine oxidase by the 8-arylhypoxanthines. At very low concentrations, the 8-arylhypoxanthines are effective inhibitors of xanthine oxidase. Consequently, the enzymatic oxidation of the 8-arylhypoxanthines (described in Chapter 4) heavily influences their resulting effective concentration. This makes it so difficult to measure the K i parameters accurately that only the 150 values are given here. The 8-arylhypoxanthines inhibit xanthine oxidase at equimolar concentrations, and the 8-arylxanthines have much higher 150 values. Therefore, because the 150 values of the 8-arylxanthines are similar to those of the 6-arylpteridin-4-ones, the 8-arylhypoxanthines are just as effective at inhibiting xanthine oxidase as the 6-arylpteridin-4-ones.Chapter 6 contains a comparison of the inhibitory properties of the 6- arylpteridin-4-ones and the 8-arylhypoxanthines with those of several other azaheterocyclic compounds. The flexibility of the active site of xanthine oxidase is confirmed. The site, which contains the molybdenum co-factor, can accommodate compounds of different sizes. Proof of this is found in the inhibition of xanthine oxidase by a small compound like allopurinol, by larger compounds like 6-arylpteridin-4-one or 8-arylhypoxanthine, and by lin -naphthoxanthine, which is twice as large as allopurinol.With these congeneric compound series, one can also study the "wall" of an enzyme's active site. The interaction of the substituents of the 6-arylpteridin-4-ones and the 8-aryl(hypo-)xanthines with the wall of xanthine oxidase's active site seems to be governed only by steric factors. This is because electronic parameters like σdo not appear in the QSAR equations. The type I and type II binding models explain the large differences between the 150 values of the 8-arylhypoxanthines and those of the 8-arylxanthines. Although these compounds are structurally similar, their aryl substituent interacts with different parts of the active site.

Xanthine oxidase (XO) is known to be involved in the mechanism of ROS and oxidants production. XO inhibitor plays role in preventing changes in purines to uric acid so uric acid levels in serum and urine can be reduced. The aim of this study was to analyze the interactions between XO and allopurinol, cucurbitacin B, morindin, or piperine by molecular docking. We obtained XO (1FIQ), allopurinol (CID135401907), cucurbitacin B (CID5281316), morindin (CID151621), and piperine (CID638024) from the database. Molecular docking was done using Hex 8.0. The docking results were visualized with Discovery Studio 3.5. The interaction of cucurbitacin B with XO and morindin with XO resulted in low docking energy, -375.08 kcal/mol and -377.4 kcal/mol. The docking energy of piperine with XO and allopurinol with XO was -163.32 kcal/mol and -281.4 kcal/mol. Cucurbitacin B and morindin bound to the active site of XO precisely on the FAD domain involving ARG426, ALA338, and ASP360. Both of the compounds established more than 10 bonds of van der waals when interacted with XO. Piperine and allopurinol bound to XO near the Fe2S cofactor. This study suggests that cucurbitacin B and morindin may have high potential as xanthine oxidase inhibitors. Keywords: allopurinol, cucurbitacine B, morindin, piperin, xanthine oxidase

In this study, the antioxidant properties of 80 % ethanol extracts of 16 species of plants from Jeju Island in Korea were evaluated using various antioxidant assays, including the DPPH (1,1-Diphenyl-2-pricrylhydrazyl) radical scavenging, superoxide scavenging, xanthine oxidase inhibition and hydrogen peroxide scavenging activities. Among the 16 plant extracts tested, CN-13 showed strong antioxidant properties in the DPPH radical scavenging and hydrogen peroxide scavenging tests. The CN-13 ethanol extract was thus selected to be used for further experiments, and was separated into various fractions using four different organic solvents (n-hexane, methylene chloride, ethyl acetate and butanol). The ethyl acetate fraction of CN-13 extract evidenced strong DPPH radical scavenging properties as compared to the other fractions. The ethyl acetate fraction also strongly inhibited DNA-damage induced by hydrogen peroxide-oxidative damage in a mouse lymphoma (L5178Y-R) cell line. Moreover, a correlation between the total phenolic content of the extract, and its antioxidant property was reported.

Investigation of the interaction between benzaldehyde thiosemicarbazone compounds and xanthine oxidase

Chalcone derivatives as xanthine oxidase inhibitors: synthesis, binding mode investigation, biological evaluation, and ADMET prediction

Kaempferia subglobosa is a perennial medicinal plant in the Zingiberaceae family, identified as a new species in January 2024. To uncover the biological benefits of K. subglobosa and its compounds, investigation of the metabolites of the roots and rhizomes, yielded three new monoterpene-chalcone conjugates, the globosones A-C, representing a rare metabolite group within the Zingiberaceae, along with six known compounds. The biogenetic pathway for the globosones involves an oxidative [3+2] cycloaddition between α-phellandrene and 4'-methoxy-4,2',6'-trihydroxychalcone. Biological testing revealed potent xanthine oxidase (XO) inhibition by globosones A and B, with IC50 values of 7.0±1.0 and 3.0±0.2 μM, respectively, surpassing the positive control drug allopurinol (IC50 7.2±0.1 μM). Globosones A-C also showed good aromatase inhibition (IC50 3.0-3.5 μM). Molecular docking studies indicated that globosones A and B may inhibit xanthine oxidase through binding at the FAD domain site. The physicochemical properties of these isolates suggest that they possess characteristics suitable for additional biological assessment in more advanced test systems. This study enhances an understanding of monoterpene-chalcone conjugate inhibitors of XO, and offers preliminary insights into the metabolites and bioactivities of K. subglobosa, uncovering potent biological activities associated with this newly discovered plant species.

Exploring the potential of novel xanthine oxidase inhibitory peptide (ACECD) derived from Skipjack tuna hydrolysates using affinity-ultrafiltration coupled with HPLC–MALDI-TOF/TOF-MS