Search Results (68)

β-glucuronidase is an important hydrolase, which plays an important role in drug metabolism, clinical diagnostics, and biotransformation. This study focuses on the heterologous expression, isolation, purification, and its enzymatic properties of β-glucuronidase CpGUS from Clostridium perfringens, as well as its application in the whole-cell transformation of unconjugated bilirubin from pig bile. A recombinant E. coli BL21(DE3)/pET-28a-CpGUS was constructed for the heterologous expression of CpGUS, with the majority of the expressed enzyme being soluble. Enzymatic analysis showed that CpGUS displayed optimal activity at pH 5.0 and 45 °C, and it rapidly lost activity at pH < 4.5. Metal ions, such as Mg²⁺ and Fe²⁺, enhanced CpGUS catalysis, while Zn²⁺, K⁺, Fe³⁺, Mn²⁺, Cu²⁺, and Na⁺ inhibited it. Notably, Cu²⁺ and Fe³⁺ can significantly inhibit β-glucuronidase, resulting in the complete loss of its activity. The results of the whole-cell transformation experiment show that when E.coli BL21(DE3)/ pET-28a-CpGUS at an OD₆₀₀ of 10 was incubated at pH 5.0, a temperature of 45 °C, and a rotation speed of 200 rpm for 12 h, the hydrolysis rate of the conjugated bilirubin in pig bile reached 81.1%, the yield of unconjugated bilirubin was 76.8%, and the purity of unconjugated bilirubin was 98.2%. The three-dimensional structure of CpGUS was predicted using AlphaFold2 (AlphaFold v2.0, DeepMind Technologise Limited, London, UK), and p-Nitrophenyl-β-D-Glucuronide (pNPG) and conjugated bilirubin were then docked to the CpGUS protein model using SWISSDOCK. The best docked conformations of the CpGUS–pNPG and CpGUS–conjugated bilirubin complex systems were simulated by independent 500 ns molecular dynamics (MD) runs with the RSFF2C force field, and the binding dynamic and catalytic mechanism of each system were obtained. The results indicated that π-π stacking, hydrogen bonding, and hydrophobic interactions between the key residue Tyr472 and the benzene ring of pNPG molecules are crucial for its catalytic process. Similarly, for the binding and catalysis of conjugated bilirubin by CpGUS, the π-π stacking and hydrogen bonding and hydrophobic interactions between the sidechains of residues Phe368 and Tyr472 and the benzene ring of conjugated bilirubin play a synergistic role during its catalytic process. Their total binding free energy (∆G_bind) values were calculated to be as high as −65.05 ± 12.66 and −86.70 ± 17.18 kJ/mol, respectively. These results suggest that CpGUS possesses high binding and catalytic hydrolysis properties for both pNPG and conjugated bilirubin. Full article

A dual-column tandem mode was used to establish the fingerprints of Sarcandrae herb from different origins, and their chemical compositions were characterized by UPLC-Q-TOF-MS/MS, which provided an experimental basis for the establishment of a rapid and efficient method for the overall quality control of Sarcandrae herba. For the first time, nine common components were identified from the Sarcandrae herba herbs of 24 origins, which were neochlorogenic acid, chlorogenic acid, 4-caffeoylquinic acid, eleutheroside B1, quercetin-3-O-β-D-glucuronide, neoastilbin, astilbin, isofraxidin, and rosmarinic acid, respectively. A total of 92 compounds were identified by liquid mass spectrometry. The quality of the Sarcandrae herb from 24 origins was analyzed by similarity evaluation, principal component analysis, and cluster analysis, and the chemical components of Sarcandrae herba were identified by UPLC-Q-TOF-MS/MS. The results showed that the overall analysis based on fingerprinting and mass spectrometry could differentiate the origins of the herbs. Full article

The petals of flowering plants should retain unique antioxidants that have not been found in the fruits, as the petals need to stay open to attract pollinators against photooxidation and devise a solution to avoid eating attacks. We reported that the yellow petals of freesia cultivars (Freesia x hybrida) accumulated original apocarotenoids, mono- and di-neapolitanosyl crocetin. Here, in the yellow petals, we discovered eight novel flavonoids by their structural determination, including four 3′-caffeoylquercetin 3,7-glycosides, one 3′-caffeoylquercetin 3-glycoside, and three 4′-caffeoylkaempferol 3,7-glycosides. The 3-carbon sugar part was a minor hexose dimer [D-glucosyl-D-glucoside or D-glucosyl-L-rhamnoside] with the β1,2-linkage, while the 7-carbon was usually O-glycosylated with D-glucose, L-rhamnose, or D-glucuronic acid. Such caffeoyl-flavonol glycosides were also present in freesia white petals, regardless of the cultivars and wild species. When dihydroflavonols, the last common precursors between flavonols and anthocyanins, switch to the flavonol route, these caffeoyl-flavonol glycosides are likely to be synthesized via quercetin or kaempferol. All the eight flavonoids exerted in vitro antioxidant activities against both lipid peroxidation and radical generation. Specifically, 3′-caffeoylquercetin 3-sophoroside and its 7-glucuronide showed superior antioxidant activity. Freesia yellow and white flowers have been utilized as edible flowers, indicating the importance of evaluating the human benefits and risks of newly identified flavonoids. Full article

Nelumbo nucifera has great value and development prospects in hypolipidemic applications. In this study, we comprehensively screened out multi–index components relevant to the quality of N. nucifera based on the hypolipidemic function of the flavonoid fraction of N. nucifera (FFN) combined with chemical characterizations. Firstly, in vitro antioxidant and cell experiments evaluated the hypolipidemic function of the FFN. Secondly, the chemical compositions of N. nucifera were identified by UPLS–MSⁿ technology. Then, the multi–index flavonoid components (rutin, hyperoside, isoquercitrin, quercetin–3–O–β–D–glucuronide, astragalin, and quercetin) were determined using a quantitative fingerprint combined with multivariate statistical data analysis. Finally, the quality of N. nucifera was scientifically evaluated by multi–index quantitative analysis combined with multivariate statistical data analysis, which was used to study the relationship between the content of flavonoid components and the overall quality. The above–mentioned research lays a material foundation for improving the quality standards of N. nucifera, providing a basis for developing functional foods to improve dyslipidemia. Full article

Phytochemical investigation of Staehelina uniflosculosa Sibth. & Sm. resulted in the isolation of twenty-two natural products: eleven sesquiterpene lactones, artemorin (1), tamirin (2), tanachin (3), reynosin (4), baynol C (5), desacetyl-β-cyclopyrethrosin (6), 1β-hydroxy-4α-methoxy-5α,7α,6β-eudesm-11(13)-en-6,12-olide (7), 1β,4α,6α-trihydroxyeudesm-11-en-8α,12-olide (8), 1β-hydroxy-arbusculin A (9), methyl-1β,4α,6α-trihydroxy-5α,7αH-eudesm-11(13)-en-12-oate (10) and methyl-1β,6α,8α-trihydroxy-5α,7αH-eudesma-4(15),11(13)-dien-12-oate (11); one lignan, pinoresinol (12); one norisoprenoid, loliolide (13); six flavonoids (four genins and two glycosides), hispidulin (14), nepetin (15), jaceosidin (16), eriodictyol (17), eriodictyol-3′-O-β-D-glucoside (18) and eriodictyol-7-O-β-D-glucuronide (19); and three phenolic derivatives (one phenolic acid and two phenolic glucosides), protocatechuic acid (20), arbutin (21) and nebrodenside A (22). From the isolated compounds, only nepetin (15) has been reported previously from the Staehelina genus and, to the best of our knowledge, it is the first time that compound (18) has been identified in Asteraceae. A number of these substances were tested for (a) inhibition of lipoxygenase and acetylocholinesterase, (b) their antioxidant activity using the DPPH (1,1-Diphenyl-2-picrylhydrazyl) method or/and (c) inhibition of lipid peroxidation. The tested components exhibited low antioxidant activity with the exception of 5 and 22, while the effectiveness of these compounds in the inhibition of acetylocholinesterase is limited. Furthermore, Molinspiration, an online computer tool, was used to determine the bioactivity ratings of the isolated secondary metabolites. The compounds’ bioactivity ratings for potential therapeutic targets were very promising. Full article

Levodopa is the mainstay of treatments for Parkinson’s disease (PD), but large heterogeneity exists in patient response. Increasing evidence implicates bile acids (BAs) involved in the pathogenesis of PD. Furthermore, BAs have also participated in drug bioavailability. However, the impact of BAs on levodopa response (LR) has not been investigated. This study evaluated the association between fecal BAs and LR. Levodopa challenge test (LCT) was conducted in 92 PD patients to assess LR. A total of 36 fecal BAs and plasma levodopa concentrations were detected using LC-MS/MS. The difference of BAs between subgroups with bottom and top 30% LR were analyzed and fecal samples from the two groups were collected for metagenomic shotgun analysis. No fecal BAs were significantly correlated with LR, except for chenodeoxycholic acid-3-β-D-glucuronide (CDCA-3-β-glucuronide, R = −0.228, p-value = 0.039). We found no significant difference in BAs between subgroups with bottom and top 30% LR. What is more, no significant changes in bacterial species composition related to bile acids metabolism or in the proportional representation of genes encoding known bile acids enzymes were observed between the groups. Overall, our data do not support an association between fecal BAs and levodopa response in PD patients. More precise macro-metabolomic approaches are needed to reveal the potential association between gut microbial interactions and the treatment effect of levodopa. Full article

Ischemic stroke is a common cerebrovascular disease with high mortality, high morbidity, and high disability. Cerebral ischemia/reperfusion injury seriously affects the quality of life of patients. Luteolin-7-O-β-d-glucuronide (LGU) is a major active flavonoid compound extracted from Ixeris sonchifolia (Bge.) Hance, a Chinese medicinal herb mainly used for the treatment of coronary heart disease, angina pectoris, cerebral infarction, etc. In the present study, the protective effect of LGU on cerebral ischemia/reperfusion injury was investigated in an oxygen–glucose deprivation/reoxygenation (OGD/R) neuronal model and a transient middle cerebral artery occlusion (tMCAO) rat model. In in vitro experiments, LGU was found to improve the OGD/R-induced decrease in neuronal viability effectively by the MTT assay. In in vivo experiments, neurological deficit scores, infarction volume rates, and brain water content rates were improved after a single intravenous administration of LGU. These findings suggest that LGU has significant protective effects on cerebral ischemia/reperfusion injury in vitro and in vivo. To further explore the potential mechanism of LGU on cerebral ischemia/reperfusion injury, we performed a series of tests. The results showed that a single administration of LGU decreased the content of EB and S100B and ameliorated the abnormal expression of tight junction proteins ZO-1 and occludin and metalloproteinase MMP-9 in the ischemic cerebral cortex of the tMCAO 24-h injury model. In addition, LGU also improved the tight junction structure between endothelial cells and the degree of basement membrane degradation and reduced the content of TNF-α and IL-1β in the brain tissue. Thereby, LGU attenuated cerebral ischemia/reperfusion injury by improving the permeability of the blood–brain barrier. The present study provides new insights into the therapeutic potential of LGU in cerebral ischemia. Full article

Luteolin-7-O-β-d-glucuronide (LGU) is a major active flavonoid glycoside compound that is extracted from Ixeris sonchifolia (Bge.) Hance, and it is a Chinese medicinal herb mainly used for the treatment of coronary heart disease, angina pectoris, cerebral infarction, etc. In the present study, the neuroprotective effect of LGU was investigated in an oxygen glucose deprivation (OGD) model and a middle cerebral artery occlusion (MCAO) rat model. In vitro, LGU was found to effectively improve the OGD-induced decrease in neuronal viability and increase in neuronal death by a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a lactate dehydrogenase (LDH) leakage rate assay, respectively. LGU was also found to inhibit OGD-induced intracellular Ca²⁺ overload, adenosine triphosphate (ATP) depletion, and mitochondrial membrane potential (MMP) decrease. By Western blotting analysis, LGU significantly inhibited the OGD-induced increase in expressions of receptor-interacting serine/threonine-protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL). Moreover, molecular docking analysis showed that LGU might bind to RIP3 more stably and firmly than the RIP3 inhibitor GSK872. Immunofluorescence combined with confocal laser analyses disclosed that LGU inhibited the aggregation of MLKL to the nucleus. Our results suggest that LGU ameliorates OGD-induced rat primary cortical neuronal injury via the regulation of the RIP3/MLKL signaling pathway in vitro. In vivo, LGU was proven, for the first time, to protect the cerebral ischemia in a rat middle cerebral artery occlusion (MCAO) model, as shown by improved neurological deficit scores, infarction volume rate, and brain water content rate. The present study provides new insights into the therapeutic potential of LGU in cerebral ischemia. Full article

To elucidate the anti-inflammatory properties and constituents of Agrimonia pilosa Ledeb. (A. pilosa), a comprehensive investigation was conducted employing activity-guided isolation. The anti-inflammatory effects were evaluated through an in vitro nitric oxide (NO) assay on lipopolysaccharide (LPS)-treated RAW 264.7 macrophage cells. Seven bio-active compounds with anti-inflammatory properties were successfully isolated from the butanol fraction and identified as follows: quercetin-7-O-β-d-rhamnoside (1), apigenin-7-O-β-d-glucopyranoside (2), kaempferol-7-O-β-d-glucopyranoside (3), quercetin (4), kaempferol (5), apigenin (6), and apigenin-7-O-β-d-glucuronide-6″-butylester (7). All isolated compounds showed strong NO inhibitory activity with IC₅₀ values ranging from 1.4 to 31 µM. Compound 6 demonstrated the most potent NO inhibition. Compound 7, a rare flavonoid, was discerned as a novel anti-inflammatory agent, ascertained through its inaugural demonstration of nitric oxide inhibition. Subsequently, a comprehensive structure-activity relationship (SAR) analysis was conducted employing eight flavonoids derived from A. pilosa. The outcomes elucidated that flavones exhibit superior NO inhibitory effects compared to flavonols, and the aglycone form manifests greater potency in NO inhibition than the glycone counterpart. These results highlight A. pilosa as a promising source of effective anti-inflammatory agents and indicate its potential as a health-beneficial dietary supplement and therapeutic material. Full article

Huangqin tea (HQT), a Non-Camellia Tea derived from the aerial parts of Scutellaria baicalensis, is widely used in the north of China. The intervention effects of HQT on intestinal inflammation and tumors have been found recently, but the active ingredient and mechanism of action remain unclear. This study aimed to investigate the interactions between the potential flavonoid active components and gut microbiota through culture experiments in vitro combined with HPLC-UV, UPLC-QTOF-MS, and 16S rDNA sequencing technology. The results showed that the HQT total flavonoids were mainly composed of isocarthamidin-7-O-β-D-glucuronide, carthamidin-7-O-β-D-glucuronide, scutellarin, and others, which interact closely with gut microbiota. After 48 h, the primary flavonoid glycosides transformed into corresponding aglycones with varying degrees of deglycosylation. The composition of the intestinal microbiota was changed significantly. The beneficial bacteria, such as Enterococcus and Parabacteroides, were promoted, while the harmful bacteria, such as Shigella, were inhibited. The functional prediction results have indicated notable regulatory effects exerted by total flavonoids and scutellarin on various pathways, including purine metabolism and aminoacyl-tRNA biosynthesis, among others, to play a role in the intervention of inflammation and tumor-related diseases. These findings provided valuable insights for further in-depth research and investigation of the active ingredients, metabolic processes, and mechanisms of HQT. Full article

D-Glucuronic acid is a fundamental building block of many biologically important polysaccharides, either in its non-substituted form or bearing a variety of substituents, among them sulfates. We have previously performed a study of the effects of exhaustive sulfation on the conformational behavior of β-gluronopyranosides. Herein, we report an investigation comparing α- and β-derivatives of this monosaccharide within the title disaccharides using NMR and quantum chemistry approaches. It was found that for α-linked disaccharides, the introduction of sulfates did not greatly affect their conformational behavior. However, for β-derivatives, considerable conformational changes were observed. In general, they resemble those that took place for the monosaccharides, except that NOESY experiments and calculations of intra-ring spin–spin coupling constants suggest the presence of a ¹S₅ conformer along with ³S₁ in the fully sulfated disaccharide. During the synthesis of model compounds, hydrogen bond-mediated aglycone delivery was used as an α-directing stereocontrol approach in the glucuronidation reaction. Full article

Eucalyptus globulus leaves contain various types of phenolic metabolites related to their antioxidant effects such as acids, catechin, flavonoids, and others. To optimize its antioxidative phenolic contents, E. globulus was extracted under various solvent conditions using 0, 10, 30, 50, 70, 90, and 100% ethanol. The 50% ethanol extract possessed the highest content of total phenolics with 497.7 mg GAE (gallic acid equivalent)/g extract. In contrast, the highest content of total flavonoids was evaluated in the 100% ethanol extract, having 169.3 mg QE (quercetin equivalent)/g extract. The antioxidant activity of various extraction conditions was assessed against the radical scavenging effect of DPPH (SC₅₀ = 188.2~5841.7 μg/mL) and ABTS (SC₅₀ = 14.2~171.3 μg/mL). The major chemical composition of E. globulus leaves was identified as including salicylic acid β-D-glucuronide (1), chlorogenic acid (2), epicatechin (3), 2″-O-galloylhyperin (4), isoquercitrin (5), isorhapontin (6), quercitrin (7), and quercetin-3-O-glucuronide (8) using LC-Q-TOF/MS analysis. Among them, the identified metabolites were clarified and their contents in the extracts were calculated via quantitative analysis using HPLC at 254 nm. The flavonoids (4, 5, 7, and 8) were determined to have an influence on the TPC, TFC, and antioxidant activity of E. globulus leaves. The results suggested that optimizing the extraction conditions can result in appropriate chemical composition and antioxidant activity. Full article

Diosmin is used to relieve chronic venous disease (CVD) symptoms. This study aimed to investigate the anti-inflammatory and antioxidant effects of diosmetin-3-O-β-d-glucuronide, the major metabolite of diosmin, using human skin explants. The explants were exposed to substance P (inflammation model) or UVB irradiation (oxidative model) and to five diosmetin-3-O-β-d-glucuronide concentrations. Inflammation was evaluated through interleukin-8 (IL-8) secretion measurements and capillary dilation observation, and oxidation was evaluated by measuring the hydrogen peroxide levels and observing cyclobutane pyrimidine dimers (CPDs). In substance-P-exposed explants, diosmetin-3-O-β-d-glucuronide induced a significant decrease in IL-8 secretions, with a maximal effect at 2700 pg/mL (−49.6%), and it reduced the proportion of dilated capillaries and the mean luminal cross-sectional area (p < 0.0001 at all tested concentrations), indicating a vasoconstrictive effect. In UVB-irradiated fragments, diosmetin-3-O-β-d-glucuronide induced a significant decrease in hydrogen peroxide production and in the number of CPD-positive cells, reaching a maximal effect at the concentration of 2700 pg/mL (−48.6% and −52.0%, respectively). Diosmetin-3-O-β-d-glucuronide induced anti-inflammatory and antioxidant responses, with the maximal effect being reached at 2700 pg/mL and corresponding to the peak plasma concentration estimated after the oral intake of 600 mg of diosmin, the daily dose usually recommended for the treatment of CVD. These ex vivo findings suggest a protective role of diosmetin-3-O-β-d-glucuronide against inflammatory and oxidative stress affecting the vascular system in CVD pathophysiology. Full article

Lamiaceae family includes various medicinal and aromatic plants used in cosmetics and pharmaceutical industries. The present study aimed to investigate in vitro the cytotoxic, photoprotective and antioxidant activities of ten Lamiaceae taxa; Melissa officinalis subsp. altissima (Sm.) Arcang., Rosmarinus officinalis L., Salvia officinalis L., Sideritis cypria Post, S. euboea Heldr., S. perfoliata L. subsp. perfoliata, S. scardica Griseb., S. sipylea Boiss., Stachys iva Griseb., and Thymus vulgaris L. The aqueous extract of Salvia officinalis was bio-guided fractionated to obtain the main bioactive metabolites, which were evaluated for the aforementioned effects and their wound-healing potential. In total, five compounds were isolated and identified through NMR spectra, namely salvianic acid A, rosmarinic acid, salvianolic acid K, luteolin-3′-O-β-D-glucuronide and hispidulin-7-O-β-D-glucuronide. All the compounds were photoprotective and non cytotoxic, while no statistically significant oxidative stress reduction was obtained. Regarding the wound-healing potential, salvianolic acid K was the most promising candidate. Overall, this study suggests photoprotective natural agents from various Lamiaceae species, widely found in Greece, and provides a better insight into Salvia officinalis and its bioactive constituents. Full article

Licorice, a natural medicine derived from the roots and rhizomes of Glycyrrhiza species, possesses a wide range of therapeutic applications, including antiviral properties. Glycyrrhizic acid (GL) and glycyrrhetinic acid (GA) are the most important active ingredients in licorice. Glycyrrhetinic acid 3-O-mono-β-d-glucuronide (GAMG) is the active metabolite of GL. GL and its metabolites have a wide range of antiviral activities against viruses, such as, the hepatitis virus, herpes virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and so on. Although their antiviral activity has been widely reported, the specific mechanism of action involving multiple links such as the virus itself, cells, and immunity are not clearly established. In this review, we will give an update on the role of GL and its metabolites as antiviral agents, and detail relevant evidence on the potential use and mechanisms of actions. Analyzing antivirals, their signaling, and the impacts of tissue and autoimmune protection may provide promising new therapeutic strategies. Full article