The Compounds of Tuna-Shredded (<i>Thunnus</i> sp.) Fortified Banana Blossom Extracts’ Antioxidant Activity and Xanthine Oxidase Enzyme Inhibition Capacity: An <i>In Vitro-In Silico</i> Study

Synthesis, enzyme inhibition and molecular docking studies of pyrazolo[1,5-a][1,3,5] triazine derivatives as potential antioxidant agents

Synthesis, molecular docking, enzyme inhibition and antioxidant potential of new 1H-benzo[d]imidazole-5-carboxamide derivatives

To assess the activity of xanthine oxidase (XO) enzyme in keratoconic corneal epithelium and to evaluate its relationship with the keratoconus (KC) severity. This prospective and randomized study included 66 eyes of 54 KC patients who received corneal collagen cross-linking treatment and 43 eyes of 32 patients who underwent photorefractive keratectomy due to their refractive error. During surgical procedures, the corneal epithelium was mechanically scraped and gathered to analyze the XO enzyme activity spectrophotometrically. The KC group was subdivided into three groups (stages 1, 2, and 3) according to the Amsler-Krumeich classification. The results were compared between the KC and the control group and in between KC subgroups. No significant differences in age and gender were found between the KC and control groups (p = 0.064 and p = 0.296, respectively). The mean XO activity levels of the KC and control groups were 173.57 ± 87.61 and 223.70 ± 99.52mIU/mg, respectively (p < 0.001). In KC group, 33 eyes were at stage 1, 19 were at stage 2, and 14 were at stage 3. No significant difference was observed between KC subgroups regarding XO activity levels (p = 0.681). In this study, our findings revealed that ultraviolet-related pro-oxidant XO enzyme may have a role in the etiopathogenesis of KC. Further studies are needed to support our result. When we started this study in 2018, we did not have a "Clinical Trials Registration." However, we have ethics committee approval (date: 21. 02. 2018/No: 22).

Skin, our first interface to the external environment, is subjected to oxidative stress caused by a variety of factors such as solar ultraviolet, infrared and visible light, environmental pollution, including ozone and particulate matters, and psychological stress. Excessive reactive species, including reactive oxygen species and reactive nitrogen species, exacerbate skin pigmentation and aging, which further lead to skin tone unevenness, pigmentary disorder, skin roughness and wrinkles. Besides these, skin microbiota are also a very important factor ensuring the proper functions of skin. While environmental factors such as UV and pollutants impact skin microbiota compositions, skin dysbiosis results in various skin conditions. In this review, we summarize the generation of oxidative stress from exogenous and endogenous sources. We further introduce current knowledge on the possible roles of oxidative stress in skin pigmentation and aging, specifically with emphasis on oxidative stress and skin pigmentation. Meanwhile, we summarize the science and rationale of using three well-known antioxidants, namely vitamin C, resveratrol and ferulic acid, in the treatment of hyperpigmentation. Finally, we discuss the strategy for preventing oxidative stress-induced skin pigmentation and aging.

Although there have been many studies on this topic, the molecular mechanism of the toxic effects of hyperammonemia on cells has not yet been fully explained. Recent studies have held oxidative stress mechanisms responsible for hyperammonemia-induced cell damage. Kidney functions are affected in diseases associated with an increase in ammonia in the blood. Our study tries to determine whether oxidative stress mechanisms are responsible for kidney damage in chronic hyperammonemia. We also investigated whether kidney damage is dependent on possible reactive oxygen products associated with the xanthine oxidase (XO) enzyme and whether the possible association can be inhibited with allopurinol, an XO enzyme inhibitor. The study took into consideration the enzyme activities of XO, xanthine dehydrogenase (XDH), superoxide dismutase (SOD), glutathione-S-transferase (GST), as well as protein thiol (P–SH) and malondialdehyde (MDA) levels. The data found demonstrated that chronic hyperammonemia had oxidative stress effects on the kidney, and that kidney XO and XDH activity changed. However, it was not possible to inhibit this oxidative stress in the kidney using allopurinol. Thus, we could not conclude that oxidative stress is an XO‐dependent mechanism. The outcomes of the study suggested that this oxidative situation arising in hyperammonemia occurred through a mechanism other than the XO enzyme.

S-allyl cysteine as potent anti-gout drug: Insight into the xanthine oxidase inhibition and anti-inflammatory activity

Luteolin, naringenin, myricetin, and ampelopsin are abundant flavonoids in nature, and several dietary supplements also contain them at very high doses. After the peroral intake, flavonoids go through extensive presystemic biotransformation; therefore, typically their sulfate/glucuronic acid conjugates reach high concentrations in the circulation. Xanthine oxidase (XO) enzyme is involved in uric acid production, and it also takes part in the elimination of certain drugs (e.g., 6-mercaptopurine). The inhibitory effects of flavonoid aglycones on XO have been widely studied; however, only limited data are available regarding their sulfate and glucuronic acid conjugates. In this study, we examined the impacts of luteolin, naringenin, myricetin, ampelopsin, and their sulfate/glucuronide derivatives on XO-catalyzed xanthine and 6-mercaptopurine oxidations employing in vitro enzyme incubation assays and molecular modeling studies. Our major results/conclusions are the following: (1) Sulfate metabolites were stronger while glucuronic acid derivatives were weaker inhibitors of XO compared to the parent flavonoids. (2) Naringenin, ampelopsin, and their metabolites were weak inhibitors of the enzyme. (3) Luteolin, myricetin, and their sulfates were highly potent inhibitors of XO, and the glucuronides of luteolin showed moderate inhibitory impacts. (4) Conjugated metabolites of luteolin and myricetin can be involved in the inhibitory effects of these flavonoids on XO enzyme.

Taraxacum mirabile Wagenitz, one of the endemic riches of Anatolia, is a species that has remained largely unexplored regarding its enzyme inhibition profile despite its pharmacological potential. The effects of T. mirabile aerial and root extracts, obtained at different polarities, were scrutinized in this study against two important enzymes: lactoperoxidase (LPO), which plays a vital role in the innate immune system, and xanthine oxidase (XO), which is prominently associated with hyperuricemia and oxidative stress. The aerial and root portions of the plant were extracted into fractions of varying polarities using petroleum ether, dichloromethane, ethyl acetate, and butanol. LPO was isolated from buffalo milk (881.6-fold purification, 22.5% yield, and 1249.9 EU/mg specific activity) via affinity chromatography and used in in vitro inhibition assays alongside commercial bovine XO enzyme. The results showed that the ethyl acetate fraction of the aerial part of the plant exhibited the strongest LPO inhibition (IC50: 15.60 ± 0.77 µg/mL) among the fractions. The petroleum ether fraction of both the aerial part (IC50: 11.17 ± 0.94 µg/mL) and the root part (IC50: 11.61 ± 0.59 µg/mL) had the highest inhibitory effect for the XO enzyme. These distinct inhibition profiles allow for significant insights into how plant extracts with varying polarities modulate XO and LPO enzymes. In conclusion, the significant inhibitory activity of T. mirabile extracts toward LPO and XO enzymes highlights their potential as a natural source for developing effective enzyme inhibitors, which could be useful for therapeutic applications.

Oxovanadium(IV) complexes with tetradentate thiosemicarbazones. Synthesis, characterization, anticancer enzyme inhibition and in vitro cytotoxicity on breast cancer cells

Xanthine oxidase is an enzyme involved in the catalysis of the oxidation reaction that converts hypoxanthine into xanthine and subsequently into uric acid. Elevated uric acid levels can pose various health risks. Gout treatment can be achieved by inhibiting the activity of the xanthine oxidase enzyme using black garlic. This study aims to identify the chemical compounds in black garlic methanol extract, assess the inhibitory effect of this extract on the xanthine oxidase enzyme using in silico methods, and determine the type of inhibition based on enzyme kinetics. The in silico analysis was conducted to evaluate the binding affinity of flavonoid compounds in black garlic extract with xanthine oxidase. The in vitro analysis tested the inhibition of the xanthine oxidase enzyme by black garlic using the UV-Vis spectrophotometry method at a wavelength of 291.7 nm, based on a decrease in uric acid concentration as an indicator of reduced enzyme activity. The type of inhibition mechanism was determined through enzyme kinetics using the Michaelis-Menten equation, which was transformed into the Lineweaver-Burk equation in a double reciprocal form. Black garlic methanol extract contains 133 chemical compounds, including 22 flavonoid compounds that are thought to inhibit xanthine oxidase. According to in silico studies, quercetin-3-O-malonylglucoside exhibits the lowest binding affinity (-9.2 kcal/mol) with the xanthine oxidase enzyme compared to the xanthine substrate (-5.2 kcal/mol) and allopurinol (-5.3 kcal/mol). Inhibition of the xanthine oxidase enzyme by black garlic demonstrated the highest inhibition of 76.352% at a concentration of 10 ppm of black garlic extract. The inhibition type of the xanthine oxidase enzyme by black garlic methanol extract showed a competitive inhibition mechanism, evidenced by an increase in the KM value from 0.014 to 0.134 without a significant change in the Vmax value. Thus, it can be concluded that black garlic extract has the potential to be a natural inhibitor of the enzyme xanthine oxidase that can be used to treat gout or hyperuricemia.

BackgroundThe disorder of uric acid metabolism is closely associated with gut microbiota and short-chain fatty acids (SCFAs) dysregulation, but the biological mechanism is unclear, limiting the development of uric acid-lowering active polysaccharides. Konjac glucomannan (KGM) could attenuate metabolic disturbance of uric acid and modulate the gut microbiota. However, the relationship between uric acid metabolism and gut microbiota is still unknown.MethodsIn this study, The fecal samples were provided by healthy volunteers and hyperuricemia (HUA) patients. Fecal samples from healthy volunteers was regarded as the NOR group. Similarly, 10% HUA fecal suspension was named as the HUA group. Then, fecal supernatant was inoculated into a growth basal medium containing glucose or KGM, and healthy fecal samples were designated as the NOR-GLU and NOR-KGM groups, while HUA fecal samples were designated as the HUA-GLU and HUA-KGM groups. All samples were cultured in an anaerobic bag system. After fermentation for 24 h, the samples were collected for further analysis of composition of intestinal microbiota, SCFAs concentration and XOD enzyme activity.ResultsThe results showed that KGM could be utilized and degraded by the gut microbiota from HUA subjects, and it could modulate the composition and structure of their HUA gut microbiota to more closely resemble that of a healthy group. In addition, KGM showed a superior modulated effect on HUA gut microbiota by increasing Megasphaera, Faecalibacterium, Lachnoclostridium, Lachnospiraceae, Anaerostipes, and Ruminococcus levels and decreasing Butyricicoccus, Eisenbergiella, and Enterococcus levels. Furthermore, the fermentation solution of KGM showed an inhibitory effect on xanthine oxidase (XOD) enzyme activity, which might be due to metabolites such as SCFAs.ConclusionIn conclusion, the effect of KGM on hyperuricemia subjects was investigated based on the gut microbiota in vitro. In the present study. It was found that KGM could be metabolized into SCFAs by HUA gut microbiota. Furthermore, KGM could modulate the structure of HUA gut microbiota. At the genus level, KGM could decrease the relative abundances of Butyricicoccus, Eisenbergiella, and Enterococcus, while Lachnoclostridium and Lachnospiraceae in HUA gut microbiota were significantly increased by the addition of KGM. The metabolites of gut microbiota, such as SCFAs, might be responsible for the inhibition of XOD activity. Thus, KGM exhibited a superior probiotic function on the HUA gut microbiota, which is expected as a promising candidate for remodeling the HUA gut microbiota.

This study investigated the phenolic profile, antioxidant capacity, and anti-gout potential of methanolic extracts obtained from flowers, leaves, and stems ofDiplotaxis harrasubsp.crassifolia. Total phenolic and flavonoid contents were quantified spectrophotometrically, and individual phenolics were characterized by LC-MS/MS, revealing 15 compounds in flowers, 11 in leaves, and 7 in stems, with quinic acid as the predominant constituent. Antioxidant activity was evaluated using DPPH, ABTS, CUPRAC, FRAP, phosphomolybdenum, and metal-chelation assays, in which flower extracts consistently exhibited the highest antioxidant potential. Xanthine oxidase (XO) enzyme inhibition assay revealed that the flower extract exhibited the best inhibition activity, with an IC50 of 0.212mg/mL, comparable to that of the positive control allopurinol (0.25mg/mL). Consistently, molecular docking showed that gentisic and protocatechuic acids, most phenolics in the extract, bind XO with more favorable energies than allopurinol through extensive hydrogen-bonding and hydrophobic interactions at key active site residues. Overall, this initial organ-specific evaluation ofD. harraprovides strong support for its flower extracts as promising candidates for standardized anti-gout preparations and as scaffolds for future XO inhibitor development.

The natural sesamol, present in the seeds and oil of sesame, has attracted considerable interest for its powerful antioxidant properties. Its ability to neutralize free radicals and to inhibit lipid peroxidation highlight its potential as a therapeutic agent to reduce the oxidative stress. This explains the beneficial effects of sesamol as effective protectors against the damaging effects of reactive oxygen species (ROS). The antioxidant activity of sesamol and its ortho mono- and di-substituted amino (R= NH2, NHMe and NHCN) derivatives was investigated at the SMD//M06-2X/6-311+G(d,p) computational level. The calculations were performed in the gas phase and in non-polar (toluene) and polar (ethanol and water) solvents. The main mechanisms, namely, HAT, SPLET and SET-PT were thoroughly investigated and analyzed. The obtained results put in evidence that HAT and SPLET are the thermodynamically favored mechanisms in non-polar and polar media respectively, while SET-PT was found a disfavored mechanism in all media. The obtained results also show that di-substitution with strong electron-donating groups in the ortho position leads to significant increase in their antioxidant activity compared to the reference molecule (sesamol). The antioxidant activity of the sesamol derivatives was also evaluated by molecular docking to explore the possible interactions of each compound with the Xanthine Oxidase (XO) enzyme which is responsible for ROS generation and the obtained results show that these derivatives exhibit high binding affinities to the active site of the XO enzyme. Finally, the studied compounds satisfy both Lipinski and Veber druglikeness properties and could be considered as good radical scavengers.

This work was undertaken to explore the phytochemical composition, antioxidant, and enzyme-inhibiting properties of Neurada procumbens L. extracts/fractions of varying polarity (methanol extract and its fractions including n-hexane, chloroform, n-butanol, and aqueous fractions). A preliminary phytochemical study of all extracts/fractions, HPLC-PDA polyphenolic quantification, and GC-MS analysis of the n-hexane fraction were used to identify the phytochemical makeup. Antioxidant (DPPH), enzyme inhibition (against xanthine oxidase, carbonic anhydrase, and urease enzymes), and antibacterial activities against seven bacterial strains were performed for biological investigation. The GC-MS analysis revealed the tentative identification of 22 distinct phytochemicals in the n-hexane fraction, the majority of which belonged to the phenol, flavonoid, sesquiterpenoid, terpene, fatty acid, sterol, and triterpenoid classes of secondary metabolites. HPLC-PDA analysis quantified syringic acid, 3-OH benzoic acid, t-ferullic acid, naringin, and epicatechin in a significant amount. All of the studied extracts/fractions displayed significant antioxidant capability, with methanol extract exhibiting the highest radical-scavenging activity, as measured by an inhibitory percentage of 81.4 ± 0.7 and an IC50 value of 1.3 ± 0.3. For enzyme inhibition experiments, the n-hexane fraction was shown to be highly potent against xanthine oxidase and urease enzymes, with respective IC50 values of 2.3 ± 0.5 and 1.1 ± 0.4 mg/mL. Similarly, the methanol extract demonstrated the strongest activity against the carbonic anhydrase enzyme, with an IC50 value of 2.2 ± 0.4 mg/mL. Moreover, all the studied extracts/fractions presented moderate antibacterial potential against seven bacterial strains. Molecular docking of the five molecules β-amyrin, campesterol, ergosta-4,6,22-trien-3β-ol, stigmasterol, and caryophyllene revealed the interaction of these ligands with the investigated enzyme (xanthine oxidase). The results of the present study suggested that the N. procumbens plant may be evaluated as a possible source of bioactive compounds with multifunctional therapeutic applications.

본 연구에서는 결명자, 오미자, 구기자, 박하, 국화, 산마, 생강, 둥글레, 감초, 오가피, 녹차, 다시마를 발효시킨 들빛 발효액으로 여러 가지 생리활성에 대하여 분석하였다. 우선, 들빛 발효액의 플라보노이드 함량과 DPPH 활성을 측정하였다. 그 결과 들빛 발효액의 총 플라보이드 함량은 <TEX>$3.4{\pm}0.5\;mg/g$</TEX>으로, DPPH법을 통해 측정한 들빛 발효액의 radical 소거능은 96%로 나타났다. 들빛 발효액의 SOD 유사활성은 29%로 나타내었고, XOase에 의해 생성된 superoxide radical 소거능은 88%로 높게 나타났다. 들빛 발효액의 항고혈압 측정실험에서는 66%의 저해률을 나타내어 ACE 저해 활성이 뛰어난 것으로 나타났다. 혈전분해능에 대한 들빛 발효액의 분석 결과는 혈전용해제로 알려져 있는 plasmin과 유사한 활성을 보이고 있다. 들빛 발효액과 면역활성과의 연관성은 NO 생성 증가율과 LPS에 의해 유도되는 NO 생성 억제율 분석으로 조사되었다. 그 결과, 들빛 발효액은 무려 15배의 높은 NO 생성 증가율을 보였다. 또한 LPS에 의해 유도된 NO 생성 억제율은 들빛 발효액 100%에서 42%로 나타나 높은 면역조절능을 나타내었다. 이상의 결과는 들빛 발효액의 우수한 생리활성을 증명하고 있고, 항산화력, 항고혈압 효과, 혈전분해능 및 면역 조절활성이 높은 것으로 나타나 기능성 음료의 소재로서 그 활용도가 높을 것으로 판단된다. This study was aimed to investigate the contents of flavonoids and the biological activity of fermented beverage of medical plants, DeulBit (DB). 50 g of Cassia semen (Cassia tora L.), 50 g of Omija (Schisandra chinensis Baillon.), 50 g of Gugija (Lycium chinense Mill), 50g of Menthae herba, 75 g of Chrysanthemum indicum Linne, 25 g of Dioscorea batatas, 5 g of Lindera obtusiloba Blume, 150 g of Polygonatum odoratum, 25 g of Glycyrrhiza uralensis, 25 g of Acanthopanacis cortex, 100 g of green tea (Camellia sinensis), and 100 g of Laminaria japonica was fermented with sucrose (<TEX>$50.0{\sim}60.0^{\circ}Brix$</TEX>.) and 0.5% of deep sea water in 10 L of distilled water for six months at room temperature. Total flavonoids contents of DB was calculated to <TEX>$3.4{\pm}0.5\;{\mu}g/g$</TEX> and antioxidative activity of DB was measured by using DPPH radical scavenging and SOD-like activity. DPPH radical scavenging and SOD-like activity of DB was 96% and 29% at 100% of DB, respectively. In addition, DB indicated about 88% and 66% of the xanthine oxidase and angiotensin converting enzyme inhibitory activities at 1% and 10% of DB, respectively and showed fibrinolytic activity. Nitric oxide (NO) synthesis was increased to 15 times by addition of DB. In addition, NO productions of the macrophages RAW264.7 cells stimulated with lipopolysaccharide (LPS) were reduced to 40.4% by addition of DB. These results suggested that DB is significant role for antioxidative and fibrinolytic activity, and have the strong xanthine oxidase and angiotensin converting enzyme inhibitory activities.