Xanthine Oxidase Inhibitory Assays Research Articles

Chemical Comparison and Identification of Xanthine Oxidase Inhibitors of Dioscoreae Hypoglaucae Rhizoma and Dioscoreae Spongiosae Rhizoma by Chemometric Analysis and Spectrum-Effect Relationship.

Dioscoreae hypoglaucae Rhizoma (DH) and Dioscoreae spongiosae Rhizoma (DS) are two similar Chinese herbal medicines derived from the Dioscorea family. DH and DS have been used as medicines in China and other Asian countries for a long time, but study on their phytochemicals and bioactive composition is limited. This present study aimed to compare the chemical compositions of DH and DS, and explore the anti-xanthine oxidase components based on chemometric analysis and spectrum-effect relationship. Firstly, an HPLC method was used to establish the chemical fingerprints of DH and DS samples, and nine common peaks were selected. Then, hierarchical clustering analysis, principal component analysis and orthogonal partial least squares discriminant analysis were employed to compare and discriminate DH and DS samples based on the fingerprints data, and four steroidal saponins compounds (protodioscin, protogracillin, dioscin, gracillin) could be chemical markers responsible for the differences between DH and DS. Meanwhile, the anti-xanthine oxidase activities of these two herbal medicines were evaluated by xanthine oxidase inhibitory assay in vitro. Pearson correlation analysis and partial least squares regression analysis were subsequently used to investigate the spectrum-effect relationship between chemical fingerprints and xanthine oxidase inhibitory activities. The results showed that four steroidal saponins, including protodioscin, protogracillin, methyl protodioscin and pseudoprogracillin could be potential anti-xanthine oxidase compounds in DH and DS. Furthermore, the xanthine oxidase inhibitory activities of the four selected inhibitors were validated by anti-xanthine oxidase inhibitory assessment and molecular docking experiments. The present work provided evidence for understanding of the chemical differences and the discovery of the anti-xanthine oxidase constituent of DH and DS, which could be useful for quality evaluation and bioactive components screening of these two herbal medicines.

Anti-Hyperuricemic Effect of Ethyl Acetate Sub-Fractions from Chrysanthemum morifolium Ramat. Dried Flowers on Potassium Oxonate-Induced Hyperuricemic Rats

Xanthine oxidase (XO) plays an important role in purine degradation in humans. The study aimed to determine the XO inhibitory potential of Chrysanthemum morifolium dried flower ethyl acetate sub-fractions and its anti-hyperuricemic effect in rat models. Bioassay-guided fractionation based on XO inhibitory assay was employed to obtain bioactive fractions and sub-fractions. In vitro cytotoxicity and cellular antioxidant capacity of the sub-fraction and its mode of XO inhibition were also investigated. The anti-hyperuricemic effect of the bioactive sub-fraction was investigated using rat models via oral consumption, and followed by an XO mRNA gene expression study. The compounds in the bioactive sub-fractions were identified putatively using HPLC-Q-TOF-MS/MS. Ethyl acetate (EtOAc) fraction exhibited the highest XO inhibition among the fractions. It was further fractionated into 15 sub-fractions. F10 exhibited high XO inhibitory activity, cellular pro-proliferative effect, and intracellular antioxidant activity among the sub-fractions tested. This sub-fraction was non-cytotoxic at 0.1–10 µg/mL, and very effective in lowering serum and urine uric acid level in rat models upon oral consumption. A total of 26 known compounds were identified and seven unknown compounds were detected via HPLC-Q-TOF–MS/MS analysis. The possible mechanisms contributing to the anti-hyperuricemic effect were suggested to be the non-competitive inhibition of XO enzyme, XO gene expression down-regulation, and the enhancement of uric acid excretion.

The influence of chemical composition of potent inhibitors in the hydrolyzed extracts of anti-hyperuricemic plants to their xanthine oxidase activities

Ethnopharmacological relevanceAnti-hyperuricemic plant parts that were selected for this study, are traditionally used to treat gout in Malaysia. Caffeic acid (a hydroxycinnamic acid), apigenin (a flavone), myricetin, quercetin and kaempferol (flavonols), were reported to act as potent xanthine oxidase inhibitors. These compounds can be found in some of the selected ethnomedicinal plants. However, there is still lack of published research works on the quantification of these inhibitors from these urate-lowering phytotherapies. Aims of the studyThe compounds were quantified from 21 hydrolyzed extracts of the phytotherapies for gout. The activity-content contributions of the compounds to the potent extracts were determined. Materials and methodsThe anti-hyperuricemic activities of the extracts and the compounds were determined using a xanthine oxidase inhibitory assay. Ultra-Performance Liquid Chromatography (UPLC) coupled with Photodiode Array Detector (PDA) was used to quantify the compounds in the extracts. ResultsThe results revealed higher activity of the hydrolyzed extracts. The hydrolyzed extract of the flower bud of Syzygium aromaticum Merr. & L.M.Perry exhibited the highest activity (EC50 = 39.58 ± 0.10 μg/mL) due to the highest content of myricetin (42,297.55 ± 159.47 μg/g). The activity-content contribution of myricetin was 7.69%. Due to the highest activity of apigenin (EC50 = 3.27 ± 0.09 μg/mL), the highest contribution of this flavone (29.96%) to the hydrolyzed extract of Orthosiphon aristatus (Blume) Miq. was observed. ConclusionThe results revealed different contents and activities of xanthine oxidase inhibitors in the hydrolyzed extracts of anti-hyperuricemic plants can play a major role to influence the activity.

Xanthine oxidase inhibitory activity of a new isocoumarin obtained from Marantodes pumilum var. pumila leaves

BackgroundIn traditional Malay medicine, Marantodes pumilum (Blume) Kuntze (family Primulaceae) is commonly used by women to treat parturition, flatulence, dysentery, dysmenorrhea, gonorrhea, and bone diseases. Preliminary screening of some Primulaceae species showed that they possess xanthine oxidase inhibitory activity. Thus, this study aimed to investigate the xanthine oxidase inhibitory activity of three varieties of M. pumilum and their phytochemical compounds.MethodDichloromethane, methanol, and water extracts of the leaves and roots of M. pumilum var. alata, M. pumilum var. pumila, and M. pumilum var. lanceolata were tested using an in vitro xanthine oxidase inhibitory assay. Bioassay-guided fractionation and isolation were carried out on the most active extract using chromatographic techniques. The structures of the isolated compounds were determined using spectroscopic techniques.ResultsThe most active dichloromethane extract of M. pumilum var. pumila leaves (IC50 = 161.6 μg/mL) yielded one new compound, 3,7-dihydroxy-5-methoxy-4,8-dimethyl-isocoumarin (1), and five known compounds, viz. ardisiaquinone A (2), maesanin (3), stigmasterol (4), tetracosane (5), and margaric acid (6). The new compound was found to be the most active xanthine oxidase inhibitor with an IC50 value of 0.66 ± 0.01 μg/mL, which was not significantly different (p > 0.05) from that of the positive control, allopurinol (IC50 = 0.24 ± 0.00 μg/mL).ConclusionThis study suggests that the new compound 3,7-dihydroxy-5-methoxy-4,8-dimethyl-isocoumarin (1), which was isolated from the dichloromethane extract of M. pumilum var. pumila leaves, could be a potential xanthine oxidase inhibitor.

Phytochemical Characterization and High In Vitro Antioxidant Properties ofHammada elegansAlgerian Extracts: a Potent Medicinal Plant

The phytochemical screening of flowers and rest aerial parts (without flowers) of localHammada elegansBotsch., performed for the first time, revealed the presence of saponins, alkaloids, flavonoids, catechin and gallic tannins, sterols and triterpenes, cardiac glycosides, and C-glycosides. However, the combined anthracene and coumarins are present only in the air rest aerial parts. Qualitative analysis of extracts by TLC confirmed the presence of these compounds in both parts ofHammada elegans. The total phenolic content was widely variable and depends on extraction solvents. The crude methanolic extract of flowers presented the highest content (2.66 ± 0.02 mg GAE/g). The in vitro antioxidant capacity ofHammada elegansextracts was assessed by DPPH, CUPRAC, iron chelating, and xanthine oxidase inhibitory assays. The DPPH assay showed an important antiradical activity of the diethyletheric fraction of the rest aerial part (EC50 = 0.46 ± 0.05 mg/ml) compared to other extracts. However, in the CUPRAC assay, the diethyletheric extract of flowers part has an interesting ability (EC50 = 2.67 ± 0.00 mg/ml) comparatively to the other extracts, but lower than those of ascorbic acid and TBHQ. The results of the chelating effect on ferrous ions show clearly that diethyletheric and acetonic extracts of both plant parts have greater chelating powers than that of the positive control. Finally, diethyletheric extract of oth parts ofHammada elegansproved to be the most active in the inhibition of xanthine oxidase activity.

Total Phenolic Content and In vitro Antioxidant Activity of Endophytic Fungi Isolated from Calotropis procera L.

Background: Endophytic fungi are microorganisms that colonize inside the plant tissues without causing any disease symptoms. Endophytic fungi isolated from medicinal plants have emerged as an interesting source for the isolation of bioactive compounds. In this study, we selected Calotropis procera, a member of the Asclepiadaceae family commonly called ‘Aak’ to evaluate the antioxidant potential of isolated endophytic fungi. </P><P> Objective: The aim of this study was to assess the total phenolic content (TPC), antioxidant capacity by using different assay and phytochemical screening of endophytic fungi isolated from Calotropis procera (leaves, stem and root). </P><P> Method: Crude ethyl acetate extracts of 20 different endophytic fungi isolated from Calotropis procera were tested for their preliminary phytoconstituents presence, TPC estimation (by Folin–Ciocalteu colorimetric assay) and antioxidant potential [1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay, hydrogen peroxide (H2O2) free radical scavenging assay, β-carotene-linoleic free radical scavenging assay, metal chelating assay and xanthine oxidase inhibitory assay]. Results: Phenols, carbohydrates, saponins, tannins, alkaloids and flavanoids were the main phytoconstituents present in the endophytic fungi. Among the endophytes, Aspergillus nomius showed the highest TPC [72.71±1.67 µg GAE (gallic acid equivalent) /mg dry weight of fungi] and antioxidant activity for DPPH free radical scavenging assay (68.86±0.19%). A high positive linear correlation was found between TPC and xanthine oxidase inhibitory assay (R2-0.890) and between TPC and DPPH free radical scavenging assay (R2-0.839). Aspergillus and Fusarium genus species showed significant antioxidant activity by the different assay. Conclusion: The present study revealed some endophytic fungi from Calotropis procera could be a potential source of novel natural antioxidant compounds.

Ocimum gratissimum Linn. Leaf extract inhibits free radical generation and suppressed inflammation in carrageenan-induced inflammation models in rats.

Ocimum gratissimum leaf is used in managing rheumatism and other inflammatory conditions. In this study, we investigated the antioxidant and anti-inflammatory effects of phenolic extract obtained by sequential methanol extraction of O. gratissimum leaves (MEOg). The methanol extract (MEOg) was obtained after sequential maceration (n-hexane, chloroform and methanol) of dried O. gratissimum leaves. The fingerprint of the extract was obtained using a high-performance liquid chromatrographic method. In vitro effects were tested by 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), nitric oxide (NO) free radical scavenging, lipoxygenase, and xanthine oxidase inhibitory assays. MEOg was studied for anti-inflammatory activity in carrageenan-induced paw edema and air pouch inflammation in rats. HPLC fingerprint of the extract shows the presence of caffeic acid, rutin, ferulic acid, apigenin, and quercetin. Antioxidant activity of MEOg revealed an IC50 value in DPPH (31.5±0.03 μg/mL) and NO assay (201.6±0.01 μg/mL), respectively. The extract demonstrated strong xanthine oxidase inhibitory and weak antilipoxygenase activities. MEOg (100 mg/kg) significantly inhibited carrageenan-induced paw edema by 43.2%. Furthermore, MEOg (50 and 100 mg/kg) significantly reduced exudate volume, leucocyte count, neutrophil infiltration, TNF-α, nitrites, myeloperoxidase, and malondialdehyde in carrageenan-induced air pouch inflammation. MEOg also elevated the glutathione levels in the inflammatory exudates. MEOg shows potential therapeutic benefits in slowing down inflammation and oxidative stress in chronic diseases, such as arthritis.

Low inhibition of alpha-glucosidase and xanthine oxidase activities of ethanol extract of Momordica charantia fruit

Objective: To evaluate the α -glucosidase and xanthine oxidase inhibitory activities of different ethanolic and aqueous extracts of Momordica charantia fruit. Materials and Methods: The M. charantia fruits were extracted using different concentrations of ethanol in water (0, 20, 40, 60, 80 and 100 %, v/v). The samples from different ethanolic and aqueous extracts of M. charantia fruit (0, 20, 40, 60 and 100 % ethanol in water [v/v] extracts) were tested for α- glucosidase and xanthine oxidase inhibitory activities. Quercetin and allopurinol were used as positive controls for α- glucosidase and xanthine oxidase inhibitory assay, respectively. The samples were also tested for phytochemicals screening such as alkaloid, phenol, steroid, tannin, coumarins and flavonoid. The significant difference was determined by one-way ANOVA test with a Tukey comparison at confidence interval of 95%. Results: Phytochemical screening showed the presence of coumarin, alkaloid, steroid and phenol in the extracts. The six different M. charantia extracts showed low inhibition on α -glucosidase and xanthine oxidase activities. Conclusions: The antidiabetic activity of the ethanolic and aqueous extracts of M. charantia fruit was not through the inhibition of α -glucosidase and xanthine oxidase.

Biological Activities of Isolated Compounds from Three Edible Malaysian Red Seaweeds, Gracilaria changii, G. manilaensis and Gracilaria sp.

Gracilaria species are red marine macroalgae that are found abundantly in Malaysia. Gracilaria changii from Morib, Selangor, G. nanilaensis and Gracilaria sp. from Gelang Patah, Johor were used in this study. Five compounds were successfully isolated and identified as hexadecanoic acid (1), cholest-5-en-3-ol (2), 2-hydroxymyristic acid (3), cholesteryl myristate (4) and 1-(4'-methoxyphenyl)-3-(2",4",6"-trihydroxyphenyl)-3-hydroxypropanone (5) based on spectral data analysis (IR, UV, GC-MS, 'H NMR, "C NMR, HMQC and HMBC). All compounds isolated were tested for cytotoxicity (MTT assay for HL-60 and MCF-7 cell lines), and antibacterial (disc diffusion method), antioxidant (DPPH free radical scavenging assay and xanthine oxidase inhibitory assay) and acetylcholinesterase inhibitory (AChE) activity (TLC bioautographic method). Compounds I and 3 exhibited strong cytotoxic activity against HL-60 and MCF-7 cell lines. Compound 5 showed high antioxidant activity in both the DPPH free radical scavenging and xanthine oxidase inhibition assays. Compound I showed positive activity for AChE inhibitory with a minimum inhibition dose of 0.625 tg sample. All compounds demonstrated antibacterial activity producing 8 to 14 mm inhibition zones. A positive control was applied to all bioassays and experiments were performed with three replicates. Results demonstrated that three edible red seaweeds are rich sources of bioactive compounds with potential application for pharmaceutical purposes.

Anti-hyperuricemia natural products from medicinal plants used by local tribes in Indonesia

Hyperuricemia, results from the overproduction or underexcretion of uric acid. During the last step of purine metabolism, xanthine oxidase (XO) catalyzes the oxidation of xanthine and hypoxanthine into uric acid [1]. XO inhibitor has the potential to be a therapeutic agent for hyperuricemia and ROS-induced diseases [2]. Allopurinol, an XO inhibitor that has been used clinically for several decades possesses some unwanted negative effects such as hepatitis, allergies and toxicity of 6-mercaptopurine [3]. In our search into natural antihyperuricemia agents, extracts of 30 medicinal plants from 19 families were evaluated by the XO inhibitory assay. Of the 30 extracts assayed, extracts from Sonneratia caseolaris (Sonneratiaceae) leaves and Zingiber purpureum (Zingiberaceae) rhizome displayed potential to inhibit the XO with activity greater than 50% at 50 mg/mL. Bioassay-guided fractionation of the leaves extract of S. caseolaris, led to the isolation of compound 1, identified as luteolin-7-O-glucoside. Simultaneously, XO inhibitory assay-guided isolation of the extract of Z. purpurea rhizome gave compound 2, identified as 3-(3,4-dimethoxyphenyl)-3,4-dimethoxystyryl-cyclohex-1-ene. Compounds 1 and 2 inhibited the activity of XO with the IC50 values of 12 mg/mL and 27 mg/mL, respectively. Allopurinol showed IC50 value of 6 mg/mL in our XO inhibitory activity assay.

In vitro Xanthine Oxidase Inhibitory Studies of Lippia nodiflora and Isolated Flavonoids and Phenylethanoid Glycosides as Potential Uric Acid-lowering Agents

Lippia nodiflora has been traditionally used for treatment of knee joint pain. Hitherto, no studies have been reported on the effective use of L. nodiflora against hyperuricemia, gout or other metabolic disorders. In this present study, L. nodiflora was examined for its ability to lower uric acid levels using an in vitro xanthine oxidase inhibitory assay. The whole plant methanolic extract was subjected to bioactivity-guided fractionation to yield 4 fractions (F1-F4). F3 displayed the highest potency and was further purified by column chromatography to afford two phenylethanoid glycosides, arenarioside (1) and verbascoside (2), and three flavonoids, 6-hydroxyluteolin (3), 6-hydroxyluteolin-7-O-glycoside (4), and nodifloretin (5). These compounds inhibited xanthine oxidase activity, with IC50 values between 7.52 ± 0.01 and 130.00 ± 2.25 μM, of which 3 was the most potent. In contrast, allopurinol, serving as a positive control, was 0.22 ± 0.00 μM. Thus, L. nodiflora, and its chemical constituents are worthy of further studies as potential anti-hyperuricemic agents.