Xanthine Oxidase Inhibitory Activity Research Articles

Effects of Processing Conditions and Simulated Digestion In Vitro on the Antioxidant Activity, Inhibition of Xanthine Oxidase and Bioaccessibility of Epicatechin Gallate.

The bioactivity and gastrointestinal stability of epicatechin gallate (ECG) may be affected by processing conditions. Results showed that the antioxidant ability and inhibitory activity on xanthine oxidase (XO) of ECG were higher at low pH values. Appropriate microwave and heating treatments improved the antioxidant (the scavenging rate increased from 71.75% to 92.71% and 80.88% under the microwave and heating treatments) and XO inhibitory activity (the inhibitory rate increased from 47.11% to 56.89% and 51.85% at the microwave and heating treatments) of ECG. The treated ECG led to a more compact structure of XO. Moreover, there may be synergistic antioxidant and inhibitory effects between ECG and its degradation products. The bioaccessibility of ECG after simulated digestion was untreated > microwave > heating, and the microwave-treated ECG still had good XO inhibitory activity after digestion. These findings may provide some significant information for the development of functional foods enriched in catechins.

Paeonia × suffruticosa Andrews leaf extract and its main component apigenin 7-O-glucoside ameliorate hyperuricemia by inhibiting xanthine oxidase activity and regulating renal urate transporters

BackgroundHyperuricemia is an important pathological basis of gout and a distinct hazard factor for metabolic syndromes and cardiovascular and chronic renal disease, but lacks safe and effective treatments currently. Paeonia × suffruticosa Andrews leaf effectively reduced serum uric acid in gout patients; however, the material foundation and the mechanism remain unclear. PurposeTo determine the primary active components and mechanism of P. suffruticosa leaf in hyperuricemic mice. MethodsThe chemical constituents of P. suffruticosa leaf was identified using high-performance liquid chromatographic analysis. The anti-hyperuricemic activity of P. suffruticosa leaf extract (12.5, 25, 50, 100, and 200 mg/kg) and its components was evaluated in hyperuricemic mice induced by a high purine diet for 14 days. Then, the urate-lowering effects of apigenin 7-O-glucoside (0.09, 0.18, and 0.36 mg/kg) were assessed in another hyperuricemic mice model built by administrating potassium oxonate and adenine for 4 weeks. The inhibitory effect of apigenin 7-O-glucoside on uric acid production was elucidated by investigating xanthine oxidase activity in vitro and in serum and the liver and through molecular docking. Immunofluorescence and western blot analyses of the expression of renal urate transporter 1 (URAT1), glucose transporter 9 (GLUT9), organic anion transporters 1 (OAT1), and ATP-binding cassette G member 2 (ABCG2) proteins elucidated how apigenin 7-O-glucoside promoted uric acid excretion. ResultsSix compounds were identified in P. suffruticosa leaf: gallic acid, methyl gallate, oxypaeoniflorin, paeoniflorin, galloylpaeoniflorin, and apigenin 7-O-glucoside. P. suffruticosa leaf extract significantly attenuated increased serum uric acid, creatinine, and xanthine oxidase activity in hyperuricemic mice. Apigenin 7-O-glucoside from P. suffruticosa leaf reduced uric acid, creatinine, and malondialdehyde serum levels, increased superoxide dismutase activity, and partially restored the spleen coefficient in hyperuricemic mice. Apigenin 7-O-glucoside inhibited xanthine oxidase activity in vitro and decreased serum and liver xanthine oxidase activity and liver xanthine oxidase protein expression in hyperuricemic mice. Molecular docking revealed that apigenin 7-O-glucoside bound to xanthine oxidase. Apigenin 7-O-glucoside facilitated uric acid excretion by modulating the renal urate transporters URAT1, GLUT9, OAT1, and ABCG2. Apigenin 7-O-glucoside protected against renal damage and oxidative stress caused by hyperuricemia by reducing serum creatinine, blood urea nitrogen, malondialdehyde, and renal reactive oxygen species levels; increasing serum and renal superoxide dismutase activity; restoring the renal coefficient; and reducing renal pathological injury. ConclusionApigenin 7-O-glucoside is the main urate-lowering active component of P. suffruticosa leaf extract in the hyperuricemic mice. It suppressed liver xanthine oxidase activity to decrease uric acid synthesis and modulated renal urate transporters to stimulate uric acid excretion, alleviating kidney damage caused by hyperuricemia.

Mechanism of flavonoids inhibiting xanthine oxidase and alleviating hyperuricemia from structure–activity relationship and animal experiments: A review

AbstractHyperuricemia (HUA) is a metabolic disease caused by excessive uric acid in patients. Flavonoids have attracted extensive attention due to their safety, effectiveness, and little side effects in the treatment of HUA and inhibition of xanthine oxidase (XO) activity. In this paper, the studies on the treatment of HUA and the inhibition of XO activity by flavonoids (including flavones, flavonols, flavanones, flavanols, isoflavonoids, anthocyanins, and chalcones) were comprehensively reviewed from the aspects of structure–activity relationship and animal experiments. Results showed that hydrogen bonds and hydrophobic interactions were the two most important forces involved in the interaction between flavonoids and XO. Flavonoids could combine with the hydrophobic cavity of XO to cause changes in XO structure and influence the action of enzyme and substrate, thereby decreasing the catalytic activity of XO. Moreover, flavonoids undergo a series of metabolic reactions in the organism, which plays a role in alleviating HUA by affecting the expression of related genes and proteins (e.g., glucose transporter 9, urate transporter 1, and organic anion transporter 1) in the kidneys, liver, and intestinal tract of the organism and changing the internal environment in organs. In addition, this paper provides new perspectives on the shortcomings of the current research, to promote the in‐depth development and application of flavonoids.

Synthesis of some new nanoparticles mixed metal complexes of febuxostat in presence of 2,2′-bipyridine: Characterization, DFT, antioxidant and molecular docking activities

New Mn(II), Fe(III), Co(II), and Ni(II) complexes with urate lowing drug febuxostat (FEB) and 2,2′-bipyridine (Bipy) were designed and depicted. Elemental analysis, molar conductance, magnetic measurements, FT-IR, UV–visible, 1H NMR, mass spectrometry (MS), X-ray diffraction (XRD) and thermal analyses (TG-DTG) studies were used to depict complexes. The FT-IR data revealed that the FEB and Bipy have two donating sites, the oxygen of the carboxylate group and the sulfur atom of the thiazole ring in FEB while, Bipy has two nitrogens of the pyridyl ring. Based on information from the magnetic and electronic spectra, an octahedral geometry was suggested for complexes. The nature of the water molecules and their thermal stability were investigated using TG-DTG analyses. By Coats-Redfern equations determined thermodynamic properties in addition to Horowitz-Metzger equations. The purpose XRD examination of compounds was used to determine the compounds (crystalline or amorphous), average crystallite size (Cs) and Crystallographic data. The bond angles, bond lengths, dipole moment, total energy and equilibrium geometry of metal complexes were investigated by DFT calculations. FEB exhibited an antioxidant action in addition to its metal complexes, with IC50 values of 0.124, 0.316, 0.635, 0.186 and 2.234 mg/mL for FEB, Mn(II), Fe(III), Co(II) and Ni(II) compounds, respectively. The molecular docking data demonstrated that the complexes have competent stabilizing scores of −6.66, −6.50, −7.18 and −7.59 kcal/mol, respectively. The data of Co(II) complex revealed the most promising potential xanthine oxidase inhibitory activity.

Artemisia selengensis Turcz. leaves extract ameliorates hyperuricemia in mice by inhibiting hepatic xanthine oxidase activity, modulating renal uric acid transporters, and improving metabolic disorders

Artemisia selengensis Turcz. (AST) is a common edible and medicinal herb with multiple health benefits. Our previous research indicated that AST leaves, by-products of AST, possess strong in vitro xanthine oxidase (XOD) inhibitory activity, making them a potential treatment for hyperuricemia (HUA). In this study, we investigated the anti-HUA effect of AST leaves extract (ASTLE) in HUA mice and explore the underlying mechanisms. Chemical analysis showed that ASTLE is rich in caffeoylquinic acids (CQAs), including four mono-CQAs (1-CQA, 3-CQA, 5-CQA, and 4-CQA) and six di-CQAs (1,3-diCQA, 1,4-diCQA, 3,4-diCQA, 1,5-diCQA, 3,5-diCQA, and 4,5-diCQA). In HUA mice, ASTLE reduced serum uric acid level and increased urine uric acid level and fraction excretion of uric acid. ASTLE also protected against liver and kidney damage, as evidenced by decreased serum levels of glutamic oxaloacetic transaminase, glutamic-pyruvic transaminase, creatinine, and blood urea nitrogen. After ASTLE intervention, the hepatic XOD activity was inhibited. Additionally, the mRNA expression of mURAT1 and mGLUT9 was down-regulated, while the mRNA expression of mABCG2, mOAT1, and mOAT3 was up-regulated. Metabolomic analysis further suggested that ASTLE mainly regulated amino acids and their metabolites, which are associated with renal uric acid excretion function. These results demonstrate that ASTLE rich in CQAs, has beneficial effects against HUA in mice by inhibiting hepatic XOD activity, modulating the mRNA expression of renal uric acid transporters, and improving metabolic disorders in HUA mice. Our findings suggest that AST leaves may be a promising natural resource for ameliorating HUA.

Corn Silk Flavonoids Ameliorate Hyperuricemia via PI3K/AKT/NF-κB Pathway.

Hyperuricemia (HUA) is a widespread metabolic disease marked by an elevated level of uric acid, and is a risk factor for premature death. The protective effect of corn silk flavonoids (CSF) against HUA and its potential mechanisms were explored. Five important apoptosis and inflammation-related signaling pathways were identified by network pharmacological analysis. The CSF exhibited significant uric acid (UA)-lowering activity in vitro by decreasing xanthine oxidase (XOD) and increasing hypoxanthine-guanine phosphoribosyl transferase levels. In a potassium oxonate-induced HUA in vivo, CSF treatment effectively inhibited XOD activity and promoted UA excretion. Furthermore, it decreased the levels of TNF-α and IL-6 and restored pathological damage. In summary, CSF is a functional food component to improve HUA by reducing inflammation and apoptosis through the down-regulating PI3K/AKT/NF-κB pathway.

Identification of 5-[5-cyano-1-(pyridin-2-ylmethyl)-1H-indole-3-carboxamido] thiazole-4-carboxylic acid as a promising dual inhibitor of urate transporter 1 and xanthine oxidase

In combination with allopurinol, tranilast is used as an urate transporter 1 (URAT1) inhibitor for the treatment of hyperuricemia, but its structure-activity relationship concerning URAT1 inhibitory activity is rarely studied. In this paper, analogs 1–30 were designed and synthesized using scaffold hopping strategy on the basis of tranilast and the privileged scaffold indole. Then, URAT1 activity was evaluated using 14C-uric acid uptake assay with HEK293-URAT1 overexpressing cells. Compared with tranilast (inhibitory rate = 44.9% at 10 μM), most compounds displayed apparent inhibitory effects, ranging from 40.0% to 81.0% at 10 μM on URAT1. Surprisingly, along with the bringing in of a cyano group at the 5-position of indole ring, compounds 26 and 28–30 exerted xanthine oxidase (XO) inhibitory activity. In particular, compound 29 presented potency on URAT1 (48.0% at 10 μM) and XO (IC50 = 1.01 μM). Molecular simulation analysis revealed that the basic structure of compound 29 had an affinity with URAT1, and XO. Furthermore, compound 29 demonstrated a significant hypouricemic effect in a potassium oxonate-induced hyperuricemia rat model at an oral dose of 10 mg/kg during in vivo tests. In summary, tranilast analog 29 was identified as a potent dual-target inhibitor of URAT1 and XO, and a promising lead compound for further investigation.

Strong inhibition of xanthine oxidase and elastase of Baccharis trimera (Less.) DC stem extract and analysis of biologically active constituents.

Introduction: In the present study, strong xanthine oxidase and elastase activities of Baccharis trimera (Less) DC stem (BT) were evaluated and active ingredients were identified to determine the possibility of using BT extract as an anti-hyperuricemia (gout) and cosmetic functional material. Methods: Hot water, 20, 40, 60, 80, and 100% ethanolic extracts of BT were prepared. The hot water extract had the highest extraction yield whereas the 100% ethanolic extract had the lowest yield. Results and discussion: Antioxidant effects were investigated based on DPPH radical scavenging activity, reducing power, and total phenolic contents. The 80% ethanolic extract showed the highest antioxidant activity. However, the 100% ethanol BT extract showed strong xanthine oxidase and elastase inhibitory activities. Functional substances were thought to be caffeic acid and luteolin. Minor active substances such as o-coumaric acid, palmitic acid, naringenin, protocatechoic acid, and linoleic acid were identified. Through this study, we firstly reported evidence that BT stem extract could be used as functional materials with anti-hyperuricemia and skin disease improving effects. BT stem extract could be used as an anti-hyperuricemia (gout) natural drug or cosmetic material. For further study, practical studies such as optimizing BT extraction and functional experiments for hyperuricemia (gout) and skin wrinkle improvement are considered necessary.

Bioactivity assays and phytochemical analysis upon Achillea filipendulina, focusing on xanthine oxidase inhibitory and antimalarial properties

BackgroundAchillea filipendulina (L.) is an important medicinal plant and an Asian species of the Asteraceae family. A. filipendulina that grows in Central and Southwest Asia. It was chosen for study with the aim of screening biologically active compounds in the essential oil of the aerial parts and the antioxidant, antimalarial and xanthine oxidase inhibitory activities of its extract. Essential oil was extracted by using Clevenger apparatus. Extraction was done by Soxhlet apparatus with solvents of n-hexane, DCM (dichloromethane) and MeOH (methanol), and then, fractionation of extracts was accomplished through various chromatographic techniques including VLC (vacuum liquid chromatography) approach and SPE (solid phase extraction) technique.ResultsApproximately 79% of A. filipendulina essential oil components were recognized by the gas chromatography mass spectrometry (GC–MS), of which monoterpenes were 63.2% the most abundant components, except for terpenes, other compounds such as fatty acids, phenols, aromatic compounds and ketones were identified, as well antimalarial activity test was done by β-hematin formation assay and xanthine oxidase inhibitory activity was accomplished by Ransod kit, then antioxidant activity was performed by DPPH (2,2-diphenyl 1-picrylhydrzyl) free radical scavenging technique. Preliminary phytochemical screening of MeOH and DCM extracts was done by TLC (thin layer chromatography) and total phenol assay. Monoterpenoids were the main component of the aerial part’s essential oil. Among different extracts, MeOH extract had significant antioxidant and xanthine oxidase inhibitory activities with RC50 (mg/mL) = 0.0707 and IC50 = 12.8741 µg/mL, respectively. Moreover DCM extract had significant antimalarial activities with IC50 (mg/mL) = 0.2511. Among the fractions, 40% and 60% Sep-Pak fraction of MeOH extract had stronger xanthine oxidase inhibitory and antioxidant activities. The 40% fraction of MeOH extract showed the strongest antioxidant and xanthine oxidase inhibitory activities, which might be related to the presence of phenolic compounds. The 80% VLC fraction of DCM extract also indicated the strong antimalarial activities.ConclusionAs this plant revealed a high content of phenols along with high antioxidant, antimalarial and xanthine oxidase inhibitory activities, it could have great medicinal value. Our results suggested the possible use of A. filipendulina in development of highly potent new pure medicinal compounds.

Preparation, Purification, and Identification of Novel Feather Keratin-Derived Peptides with Antioxidative and Xanthine Oxidase Inhibitory Activities.

Feather keratin is an underappreciated protein resource of high quality, with limited bioavailability, and it urgently requires eco-friendly methods to enhance its value. Here, we report on the preparation, purification, and identification of novel peptides with antioxidant and xanthine oxidase (XOD) inhibitory activities from fermented feather broth, using Bacillus licheniformis 8-4. Two peptides, namely, DLCRPCGPTPLA (DA-12) and ANSCNEPCVR (AR-10), displayed remarkable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging abilities with half-maximal inhibitory concentrations (IC50) values of 0.048, 0.034, and 0.95, 0.84 mg/mL, respectively. These values exceed those of the previously reported feather keratin-derived antioxidant peptides. Another peptide, GNQQVHLQSQDM (GM-12), demonstrated XOD activity inhibition, with an IC50 value of 12.15 mg/mL, and it quenched the fluorescence of XOD. Furthermore, after simulating gastrointestinal digestion, DA-12, AR-10, and GM-12 retained their biological activities. Meanwhile, DA-12 and GM-12 showed an unexpected synergistic inhibition on XOD activity accompanied by fluorescence quenching. This study provides new insights into the potential applications of feather keratin, including functionalized feed with antioxidative and antigout (anti-hyperuricemia) activities.

Xanthine oxidase inhibitory kinetics and mechanism of ellagic acid: In vitro, in silico and in vivo studies.

Ellagic acid (EA), which is widely distributed in many foods, has been found to possess inhibitory activity against xanthine oxidase (XO). However, there is ongoing debate about the difference in XO inhibitory activity between EA and allopurinol. Additionally, the inhibitory kinetics and mechanism of EA on XO are still unclear. Herein, the authors systematically studied the inhibitory effects of EA on XO. The authors' findings showed that EA is a reversible inhibitor with mixed-type inhibition, and its inhibitory activity is weaker than allopurinol. Fluorescence quenching experiments suggested that the generation of EA-XO complex was exothermic and spontaneous. In silico analysis further confirmed that EA entered the XO catalytic centre. Furthermore, the authors verified the anti-hyperuricemia effect of EA in vivo. This study elucidates the inhibition kinetics and mechanism of EA on XO, and lays a theoretical foundation for the further development of drugs and functional foods containing EA for the treatment of hyperuricemia.

In vitro and In silico Xanthine Oxidase Inhibitory Activities of 3-Aryl-2- thioxo-2,3-dihydroquinazolin-4(1H)-one Derivatives.

Hyperuricemia is associated with several disease conditions, such as atherosclerosis, arthritis, kidney stones, and many others. Xanthine oxidase (XO) is an enzyme that catalyzes the conversion of xanthine to uric acid. Hence, XO is a major therapeutic drug target in the treatment of hyperuricemia and associated disorders. The current study aimed to identify XO inhibitors based on quinazoline derivatives, with the potential to be used against gout and other hyperuricemia-associated diseases. In the current study, eighteen quinazoline derivatives 2-19 were synthesized and assessed for their in vitro xanthine Oxidase (XO) inhibitory activity. Furthermore, the most active compounds, 5 and 17, were subjected to kinetics studies, followed by computational docking. Human BJ fibroblast cells were used to measure the cytotoxicity of active compounds. Compounds 4-6, 8, 10, 13, 15-17, and 19 were found active against XO, with an IC50 values between 33.688 to 362.173μM. The obtained results showed that compounds 5 and 17 possess a significant xanthine oxidase inhibitory activity. The kinetics and molecular docking studies suggested that compounds 5 (IC50 = 39.904 ± 0.21 μM) and 17 (IC50 = 33.688 ± 0.30 μM) bind in the allosteric site of XO and exhibit a non-competitive type of inhibition. The molecular docking studies also predicted that the NH group of the pyrimidine ring binds with Ser344 residues of XO. Furthermore, all active compounds were non-cytotoxic on the human BJ fibroblasts cell line. This study identifies a series of quinazoline compounds as xanthine oxidase inhibitors, with the potential to be further investigated.

Metabolite profiling, in vitro antioxidant and xanthine oxidase inhibitory properties of six Sumatran sidaguri (Sida L.)

Context: The Sida genus, part of the Malvaceae family, is commonly used in traditional medicine due to its many beneficial properties, such as antigout, anti-rheumatic, anti-inflammatory, antitumor, and hepatoprotective effects. Various species of Sida are found in Indonesia, particularly on the island of Sumatra, and may hold potential for future drug development. Aims: To assess and compare the methanol leaf extracts of three Sida species (S. acuta, S. rhombifolia, and S. cordifolia) from West Sumatra and South Sumatra, Indonesia, for their chemical content in metabolite profiling, in vitro antioxidant capacity, and xanthine oxidase inhibitory activity. Methods: The LC-MS/MS method was used to determine metabolite profiling, while spectrophotometry was used to assess total phenolic content, flavonoid content, antioxidant capacity, and xanthine oxidase inhibitor activity. Results: The results showed that S. rhombifolia (WS) had the highest total phenolic (64.872 ± 1.525 mg GAE/g) and flavonoid contents (13.890 ± 0.816 mg QE/g), S. acuta (SS) exhibited the highest antioxidant activity (IC50 147.295 ± 3.364 µg/mL), and S. cordifolia (WS) showed the highest xanthine oxidase inhibitor activity (IC50 110.087 ± 15.637 µg/mL). Conclusions: These findings suggest that S. acuta, S. rhombifolia, and S. cordifolia may be potential sources of polyphenols and antioxidants and could also serve as promising natural plant sources for developing antigout remedies.

Bioactivity assays and phytochemical analysis upon Alcea glabrata; focusing on xanthine oxidase inhibitory and antimalarial properties

Alcea glabrata from the family Malvaceae, was selected for evaluating its xanthine oxidase inhibitory, anti-malarial, and antioxidant activities. In addition, some phytochemical analysis upon different extracts of A. glabrata were performed. Aerial parts of the collected A. glabrata plant material were dried and solvent extracted via soxhlet apparatus using different solvents. Various chromatographic techniques were used for extra fractionation of the achieved extracts. Xanthine oxidase (XO) inhibitory, antimalarial and antioxidant activity assays upon different A. glabrata extracts and fractions were carried out and reported in terms of IC50s. Total phenolic and flavonoid contents of the A. glabrata methanol extract (MeOH) were determined using the 2,2-Di Phenyl-1-Picryl Hydrazyl (DPPH) assay, aluminum chloride colorimetric, and Folin-Ciocalteu reagents, respectively. In addition, A. glabrata essential oil was obtained through hydrodistillation by a Clevenger apparatus. Analysis and identification of essential oil compounds were carried out through gas chromatography mass spectrometry (GC-MS) analysis. MeOH extract showed the highest XO inhibitory activity with the IC50 of 0.37 ± 0.12 mg/mL antioxidant activity with the RC50 of 0.24 ± 0.06 mg/mL. While, chloroform extract revealed the strongest antimalarial activity with the IC50 of 0.4 ± 0.05 mg/mL. The total flavonoid and phenolic contents of the A. glabrata methanol extract were 39.8 mg quercetin equivalent and 6.1 g gallic acid equivalent per 100 g of dry plant material, respectively. GC-MS analysis showed that the monoterpenes were prevailing in A. glabrata essential oil where the major constituents: octacosane (30.7%), eugenol (12.3%), and anethole (12.0%). Concerning the results of this study, A. glabrata extracts and its ingredients could be considered as a novel promising herbal medicine in the design and also treatment of new drugs for the relief of gout and malaria diseases.

Purification and identification of xanthine oxidase inhibitory peptides from enzymatic hydrolysate of α-lactalbumin and bovine colostrum casein

The development of food-derived Xanthine Oxidase (XO) inhibitors is critical to the treatment of hyperuricemia and oxidative stress-related disease. Few studies report on milk protein hydrolysates’ XO inhibitory activity, with the mechanism of their interaction remaining elusive. Here, different commercial enzymes were used to hydrolyze α-lactalbumin and bovine colostrum casein. The two proteins hydrolyzed by alkaline protease exhibited the most potent XO inhibitory activity (bovine casein: IC50 = 0.13 mg mL−1; α-lactalbumin: IC50 = 0.28 mg mL−1). Eight potential XO inhibitory peptides including VYPFPGPI, GPVRGPFPIIV, VYPFPGPIPN, VYPFPGPIHN, QLKRFSFRSFIWR, LVYPFPGPIHN, AVFPSIVGR, and GFININSLR (IC50 of 4.67–8.02 mM) were purified and identified from alkaline protease hydrolysates by using gel filtration, LC-MS/MS and PeptideRanker. The most important role of inhibiting activity of peptides is linked to hydrophobic interactions and hydrogen bonding based on the results of molecular docking and molecular dynamics simulation. The enzymatic hydrolysate of α-lactalbumin and bovine colostrum casein could be a competitive candidates for hyperuricemia-resisting functional food.

Antioxidant and Immune Stimulating Effects of Allium cepa Skin in the RAW 264.7 Cells and in the C57BL/6 Mouse Immunosuppressed by Cyclophosphamide.

Allium cepa L. (onion) has been reported to have various pharmacological effects, such as preventing heart disease, and improving antimicrobial activity and immunological effects. The Republic of Korea produced 1,195,563 tons of onions (2022). The flesh of onion is used as food while the onion skin (OS) is thrown away as an agro-food by-product and is considered to induce environmental pollution. Thus, we hypothesize that increasing usage of OS as functional food material could help protect from the environment pollution. The antioxidant effects and immune-enhancing effects of OS were evaluated as functional activities of OS. In this study, OS showed high 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities and xanthine oxidase (XO) inhibitory activity. The antioxidant activities increased in a dose-dependent manner. The IC50 values of DPPH, ABTS radical scavenging activity, and XO inhibitory activity were 954.9 μg/mL, 28.0 μg/mL, and 10.7 μg/mL, respectively. Superoxide dismutase and catalase activities of OS in RAW 264.7 cells were higher than those of the media control. There was no cytotoxicity of OS found in RAW 264.7 cells. Nitric oxide and cytokines (IL-1β, IL-6, IFN-γ, and TNF-α) concentrations in RAW 264.7 cells significantly increased in a dose dependent manner. Immune-stimulating effects of OS were evaluated in immunosuppressed mice induced by cyclophosphamide. White blood cell count and the B cell proliferation of splenocytes were higher in OS100 (OS extract 100 mg/kg body weight) and OS200 (OS extract 200 mg/kg body weight) groups than in the negative control (NC) group. Serum IgG and cytokine (IL-1β and IFN-γ) levels were also higher in OS100 and OS200 groups than in the NC group. OS treatment increased NK cell activity compared with the NC group. The results suggested that OS can improve antioxidant and immune stimulating effects. The use of OS as functional supplement can reduce the agro-food by-product and it may contribute to carbon neutrality.

Releasing Bioactive Compounds from Brown Seaweed with Novel Cold-Adapted Alginate Lyase and Alcalase.

Seaweeds are considered to be third-generation renewable biomasses, the comprehensive utilization of which has drawn increasing attention in recent years. A novel cold-active alginate lyase (VfAly7) was identified from Vibrio fortis and biochemically characterized for brown seaweed utilization. The alginate lyase gene was high-level expressed in Pichia pastoris, with an enzyme yield of 560 U/mL and a protein content of 9.8 mg/mL by high-cell density fermentation. The recombinant enzyme was most active at 30 °C and pH 7.5, respectively. VfAly7 was a bifunctional alginate lyase with both poly-guluronate and poly-mannuronate hydrolysis activities. On the basis of VfAly7, a bioconversion strategy for the utilization of brown seaweed (Undaria pinnatifida) was developed. The obtained AOSs showed stronger prebiotic activity towards tested probiotics when compared to that of commercial fructooligosaccharides (FOSs), while the obtained protein hydrolysates displayed strong xanthine oxidase inhibitory activity with IC50 of 3.3 mg/mL. This study provided a novel alginate lyase tool as well as a biotransformation route for the utilization of seaweeds.

Identification of the Constituents of Ethyl Acetate Fraction from Smilax china L. and Determination of Xanthine Oxidase Inhibitory Properties

The aim of this work was to investigate the xanthine oxidase (XO)-inhibitory activity of ethanol extracts from Smilax china L. and to identify the active compounds in the ethyl acetate (EtOAc) fraction. Extraction of ethanol extracts from Smilax china L. and then ethanol extracts were concentrated, and the polyphenolic compounds were extracted with petroleum ether (PE), chloroform, EtOAc, n-butanol (n-BuOH), and residual ethanol fractions. Their effects on XO activity were then compared separately. The polyphenolic components of the EtOAc fraction were identified by HPLC and HPLC-mass spectrometry (HPLC-MS) analysis. Kinetic analysis demonstrated that all these extracts showed XO-inhibitory properties, and among them the EtOAc fraction had the strongest inhibitory effect (IC50 = 101.04 μg/mL). The inhibitory constant (Ki) of the EtOAc fraction on XO activity was 65.20 μg/mL, showing excellent inhibition on XO in the competitive mode. Sixteen compounds were identified from the EtOAc fraction. The study demonstrates that the EtOAc fraction of Smilax china L. may be a potential functional food to inhibit XO activity.

Stigmastane-type steroid saponins from the leaves of Vernonia amygdalina and their α-glucosidase and xanthine oxidase inhibitory activities

Two new vernonioside K (1) and vernonioside L (2) and four known Δ7,9(11) stigmastane-type steroidal saponins—vernonioside B2 (3), vernoniacum B (4), vernonioside B1 (5), and vernoamyoside A (6)—were isolated from the leaves of Vernonia amygdalina. Their structures were determined by comprehensive spectroscopic analysis with one-dimensional nuclear magnetic resonance, two-dimensional nuclear magnetic resonance, and high-resolution mass spectrometry. All isolated compounds (1–6) were evaluated to determine their inhibitory effects on α-glucosidase and xanthine oxidase. Among them, two new compounds 1 and 2 showed significant inhibition of α-glucosidase with IC50 values of 78.56 ± 7.28 and 14.74 ± 1.57 (μM), respectively, comparable with acarbose as a positive control (127.53 ± 1.73 μM); none of these compounds inhibited xanthine oxidase activity. Compounds 1 and 2 are promising candidates for the development of antidiabetic agents from natural sources.

UJI AKTIVITAS PENGHAMBATAN XANTIN OKSIDASE OLEH EKSTRAK ETANOL, PARTISI (KLOROFORM DAN METANOL) DAUN KUMIS KUCING (Orthosiphon stamineus Benth)

Cat's Whisker Leaf (Orthosiphon stamineus Benth) is one part of the plant that can grow well in places that are exposed to a lot of sunlight. This plant contains secondary metabolites, namely flavonoids, alkaloids, saponins, steroids, and terpenoids. These compounds have the function of being able to inhibit the xanthine oxidase enzyme associated with gout. It aims to determine the effect of xanthine oxidase inhibitory activity by ethanol extract, partition (chloroform & methanol) of cat whiskers (Orthosiphon stamineus Benth) leaves. Since it is experimental study, it used xanthine oxidase inhibition reaction method by ethanol extract, partition (chloroform & methanol) whose absorbance was measured by UV-Vis spectrophotometry and compared with the positive control, namely allopurinol. The results of the xanthine oxidase inhibition test obtained in this study were ethanol extract 50 g/mL (40.22%) 100 g/mL (52.76%), methanol partition concentration of 50 g/mL (27.30%) 100 g/ mL (43.91%), chloroform partition 50 g/mL (20.66%) 100 g/mL (41.69%) and allopurinol concentration 5 g/mL (40.95%) 10 g/mL (80, 81%). Data on the inhibition of the xanthine oxidase enzyme were analyzed using One Way ANOVA with a significant value of 0.000 and continued with the Tukey test with a test value of 100 ppm methanol partitioning inhibition having the same activity as 5 ppm allopurinol. So it can be concluded that ethanol extract, chloroform partitioning and methanol partitioning of cat whiskers leaves can inhibit xanthine oxidase. Keywords: cat whiskers leaves, xantin oksidase, spektrofotometri uv-vi