Assessment on malvidin-3-glucoside interaction with TLR4 via multi-spectroscopic analysis and molecular docking.

As one innate immune pattern recognition receptor, Toll-like receptor 4 (TLR4) recently has been considered as a critical player in glucolipid metabolism. Blueberries contain high level of anthocyanins, especially malvidin-3-glucoside (Mv-3-glc), which contribute the anti-inflammatory, hypoglycemic, and hypolipidemic effects. It is speculated that Mv-3-glc is able to possess these functions by binding to TLR4. Here, the noncovalent interactions of Mv-3-glc and TLR4 was explored through multi-techniques including fluorescence and ultraviolet-visible (UV-Vis) absorption spectroscopy, as well as molecular docking. The results demonstrated that Mv-3-glc was able to quench TLR4 intrinsic fluorescence effectively. A stable complex was formed spontaneously and the reaction was exothermic. The degree of binding of Mv-3-glc to TLR4 showed a strong dependence on the chemical concentration, temperature, and pH values. The negative signs for enthalpy (ΔH = -69.1 ± 10.8 kJ/mol) and entropy (ΔS = -105.0 ± 12.3 J/mol/K) from the interaction of the Mv-3-glc and TLR4 shows that the major driving forces are the hydrogen bonding and van der Waals' force, which is consistent with the molecular docking results. In addition, molecular docking predicted that the active center with specific amino acid residues, Phe126, Ser127, Leu54, Ile153, and Tyr131 was responsible for the site of Mv-3-glc binding to TLR4/myeloid differentiation protein-2 (MD-2). These findings confirmed that Mv-3-glc could bind to TLR4, which would be beneficial to understand the target therapeutic effects of blueberry anthocyanins on TLR4 in regulating glucolipid metabolism.

Engineering Escherichia coli for efficient and economic production of C-glycosylflavonoids by deleting YhhW and regulating pH.

C-glycosylflavonoids have a number of pharmacological activities. An efficient method for the preparation of C-glycosylflavonoids is through metabolic engineering. Thus, it is important to prevent the degradation of C-glycosylflavonoids for producing C-glycosylflavonoids in the recombinant strain. In this study, two critical factors for the degradation of C-glycosylflavonoids were clarified. The quercetinase (YhhW) gene from Escherichia coli BL21(DE3) was expressed, purified, and characterized. YhhW effectively degraded quercetin 8-C-glucoside, orientin, and isoorientin, while the degradation of vitexin and isovitexin was not significant. Zn2+ can significantly reduce the degradation of C-glycosylflavonoids by inhibiting the activity of YhhW. pH was another key factor causing the degradation of C-glycosylflavonoids, and C-glycosylflavonoids were significantly degraded with pH exceeding 7.5 in vitro or in vivo. On this basis, two strategies, deleting YhhW gene from the genome of E. coli and regulating pH during the bioconversion, were developed to relieve the degradation of C-glycosylflavonoids. Finally, the total degradation rates for orientin and quercetin 8-C-glucoside decreased from 100 to 28% and 65% to 18%, respectively. The maximum yield of orientin reached 3353 mg/L with luteolin as substrate, and the maximum yield of quercetin 8-C-glucoside reached 2236 mg/L with quercetin as substrate. Therefore, the method described herein for relieving the degradation of C-glycosylflavonoids may be widely used for the biosynthesis of C-glycosylflavonoids in recombinant strains.

LC-DAD-ESI-MS/MS-based assessment of the bioactive compounds in fresh and fermented caper (Capparis spinosa) buds and berries.

The main objective of this study was to compare bioactive compounds and other important quality parameters of fresh and fermented caper buds and berries. Fresh samples were fermented using dry-salted and brined techniques. The higher phenolic content was determined in the fresh (1843.71 mg/100 g DW) and fermented buds (1198.54-1539.49 mg/100 g DW) rather than the berries (29.72-40.75 mg/100 g DW). Quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, and quercetin-O-galloly-O-hexoside were the principal phenolic components in fresh and fermented buds while quercetin-3-O-rutinoside in fresh and fermented berries. The amounts of isorhamnetin, quercetin, and kaempferol increased in fermented buds and berries compared to fresh samples. Similarly, antioxidant capacity of buds was found to be markedly higher than berries. As for sugar compounds, it was found that fructose in buds (1.56-3.23 g/100 g DW) and glucose in berries (1.96-6.38 g/100 g DW) had the highest amount. When total phenolics and antioxidant properties were evaluated, it was observed that they were better preserved in the dry-salted samples than the brined samples.

Suppression of hTAS2R16 Signaling by Umami Substances.

Interaction between umami and bitter taste has long been observed in human sensory studies and in neural responses in animal models, however, the molecular mechanism for their action has not been delineated. Humans detect diverse bitter compounds using 25-30 members of the type 2 taste receptor (TAS2R) family of G protein-coupled receptor. In this study, we investigated the putative mechanism of antagonism by umami substances using HEK293T cells expressing hTAS2R16 and two known probenecid-insensitive mutant receptors, hTAS2R16 N96T and P44T. In wild type receptor, Glu-Glu, inosine monophosphate (IMP), and l-theanine behave as partial insurmountable antagonists, and monosodium glutamate (MSG) acts as a surmountable antagonist in comparison with probenecid as a full insurmountable antagonist. The synergism with IMP of umami substances still stands in the suppression of hTAS2R16 signaling. In mutagenesis analysis, we found that Glu-Glu, MSG, and l-theanine share at least one critical binding site on N96 and P44 with probenecid. These results provide the first evidence for a direct binding of umami substances to the hTAS2R16 through the probenecid binding pocket on the receptor, resulting in the suppression of bitterness.

Taste compound - Nanocellulose interaction assessment by fluorescence indicator displacement assay.

Interactions between taste compounds and nanofibrillar cellulose were studied. For this, a new fluorescent indicator displacement method was developed. Two fluorescent indicators, namely, Calcofluor white and Congo red, were chosen because of their specific binding to cellulose and intrinsic fluorescence. Seven taste compounds with different structures were successfully measured together with nanofibrillar cellulose (NFC) and ranked according to their binding constants. The most pronounced interactions were found between quinine and NFC (1.4 × 104 M-1), whereas sucrose, aspartame and glutamic acid did not bind at all. Naringin showed moderate binding while stevioside and caffeine exhibited low binding. The comparison with microcrystalline cellulose indicates that the larger surface area of nanofibrillated cellulose enables stronger binding between the binder and macromolecules. The developed method can be further utilized to study interactions of different compound classes with nanocellulose materials in food, pharmaceutical and dye applications, using a conventional plate reader in a high-throughput manner.

Safety Assessment of Methyl Glucose Polyethers and Esters as Used in Cosmetics.

The Cosmetic Ingredient Review Expert Panel (Panel) reviewed the safety of methyl glucose polyethers and esters which function in cosmetics as skin/hair-conditioning agents, surfactants, or viscosity increasing agents. The esters included in this assessment are mono-, di-, or tricarboxyester substituted methyl glucosides, and the polyethers are mixtures of various chain lengths. The Panel reviewed available animal and clinical data, including the molecular weights, log Kows, and other properties in making its determination of safety on these ingredients. Where there were data gaps, similarities between molecular structures, physicochemical and biological characteristics, and functions and concentrations in cosmetics allowed for extrapolation of the available toxicological data to assess the safety of the entire group. The Panel concluded that there likely would be no significant systemic exposure from cosmetic use of these ingredients, and that these ingredients are safe in cosmetic formulations in the present practices of use and concentration.

Influence of olive leaf processing on the bioaccessibility of bioactive polyphenols.

Olive leaves are rich in bioactive compounds, which are beneficial for humans. The objective of this work was to assess the influence of processing conditions (drying and extraction) of olive leaves on the extract's bioaccessibility. Thus, extracts obtained from dried olive leaves (hot air drying at 70 and 120 °C or freeze-drying) by means of conventional or ultrasound-assisted extraction were subjected to in vitro digestion. Antioxidant capacity, total phenolic content, and HPLC-DAD/MS/MS analysis were carried out during digestion. The dehydration treatment used for the olive leaves did not have a meaningful influence on bioaccessibility. The digestion process significantly (p<0.05) affected the composition of the extracts. Oleuropein and verbascoside were quite resistant to gastric digestion but were largely degraded in the intestinal phase. Nevertheless, luteolin-7-O-glucoside was the most stable polyphenol during the in vitro simulation (43% bioaccessibility). Therefore, this compound may be taken into consideration in further studies that focus on the bioactivity of olive leaf extracts.

Characterization of the beta-D-glucopyranoside binding site of the human bitter taste receptor hTAS2R16.

G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and beta-D-glucopyranoside and will also facilitate rational design of bitter blockers.

The human bitter taste receptor hTAS2R50 is activated by the two natural bitter terpenoids andrographolide and amarogentin.

Bitterness perception in mammals is mediated through activation of dedicated bitter taste receptors located in the oral cavity. Genomic analyses revealed the existence of orthologous mammalian bitter taste receptor genes, which presumably recognize the same compounds in different species, as well as species-specific receptor gene expansions believed to fulfill a critical role during evolution. In man, 8 of the 25 bitter taste receptors (hTAS2Rs) are closely related members of such an expanded subfamily of receptor genes. This study identified two natural bitter terpenoids, andrographolide and amarogentin, that are agonists for the orphan receptor hTAS2R50, the most distant member of the subfamily. This paper presents the pharmacological characterization of this receptor and analyzes its functional relationship with the previously deorphaned hTAS2R43, hTAS2R44, hTAS2R46, and hTAS2R47. Insights into the general breadth of tuning, functional redundancies, and relationships between pharmacological activation patterns and amino acid homologies for this receptor subfamily are presented.

Characterization of limonin glucoside metabolites from human prostate cell culture medium using high-performance liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry.

The metabolism of limonin 17-beta-D-glucopyranoside (LG) by non-cancerous (RWPE-1) and cancerous (PC-3) human prostate epithelial cells was investigated using high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with in-source fragmentation and tandem mass spectrometry (MS/MS). During positive ion LC/ESI-MS, LG formed an abundant sodiated species ([M+Na]+) while the protonated molecule was barely observable. [M+Na]+ further fragmented into the less abundant [LARL+H]+ and a predominantly protonated aglycone molecule (limonin) due to in-source fragmentation. The major metabolite, limonin A-ring lactone (LARL), formed an abundant protonated molecule that was fragmented into a protonated molecule of limonin by loss of one molecule of water. In MS/MS by collisionally activated dissociation (CAD), LG produced the sodiated aglycone, [aglycone+Na]+, while LARL fragmented into [M+H]+ of limonin and fragment ions resulted by further loss of water, carbon monoxide and carbon dioxide, indicating the presence of oxygenated-ring structures. The limits of detection of LG were 0.4 and 20 fmol in selected-ion monitoring (SIM) and selected-reaction monitoring (SRM) detection, respectively.

Interaction of a novel GDP exchange inhibitor with the Ras protein.

Mutated, tumorigenic Ras is present in a variety of human tumors. Compounds that inhibit tumorigenic Ras function may be useful in the treatment of Ras-related tumors. The interaction of a novel GDP exchange inhibitor (SCH-54292) with the Ras-GDP protein was studied by NMR spectroscopy. The binding of the inhibitor to the Ras protein was enhanced at low Mg2+ concentrations, which enabled the preparation of a stable complex for NMR study. To understand the enhanced inhibitor binding and the increased GDP dissociation rates of the Ras protein, the conformational changes of the Ras protein at low Mg2+ concentrations was investigated using two-dimensional 1H-15N HSQC experiments. The Ras protein existed in two conformations in slow exchange on the NMR time scale under such conditions. The conformational changes mainly occurred in the GDP binding pocket, in the switch I and the switch II regions, and were reversible. The Ras protein resumed its regular conformation after an excess amount of Mg2+ was added. A model of the inhibitor in complex with the Ras-GDP protein was derived from intra- and intermolecular NOE distance constraints, and revealed that the inhibitor bound to the critical switch II region of the Ras protein.

In-vivo assessment of extrahepatic metabolism of paeoniflorin in rats: relevance to intestinal floral metabolism.

The extraction ratios of paeoniflorin in gut wall (EG), liver (EH) and lung (EL) were assessed by comparing AUCs after various routes of its administration to estimate the first-pass effects and the metabolism by intestinal flora. Pulmonary extraction ratio of paeniflorin was assessed by comparing AUCs calculated from venous and arterial plasma concentrations after its intravenous administration (0.5 mg kg-1). The mean pulmonary extraction ratio was estimated to be 0.06. The hepatic extraction ratio (EH was assessed by comparing AUCs after intraportal and intravenous administrations (0.5 and 5 mg kg-1). The plasma concentration profiles of paeoniflorin after intraportal administration were very close to those after intravenous administration, suggesting a negligible hepatic extraction ratio of paeoniflorin. The AUC value after intraperitoneal administration (0.5 mg kg-1) was greater than that after intraportal or intravenous administration. This finding suggests that paeoniflorin is not metabolized in the gut wall. The transference of paeoniflorin from the serosal side to the mucosal side was evaluated by the in-vitro everted sac method. The low intestinal permeability (19.4% at 60 min) was demonstrated by the comparison with phenobarbital (63.1% at 60 min). We conclude that paeoniflorin is not metabolize by gut wall, liver and lung, its poor absorption from the intestine results in extremely low bioavailability and the unabsorbed fraction of paeoniflorin is degraded by the intestinal flora.

Rapid identification of Streptococcus bovis by using combination constitutive enzyme substrate hydrolyses.

Several studies have documented the association of blood and rectal-culture positivity for Streptococcus bovis with gastrointestinal neoplasia, especially colonic carcinoma. Conventional methods using bile-esculin hydrolysis, salt tolerance, and sugar fermentations to differentiate S. bovis from other streptococci are laborious, slow, and relatively expensive. Commercially available systems are costly and require at least 24 to 48 h of incubation. A rapid identification procedure for S. bovis and related bacteria was developed. The method uses a reagent containing two hydrolyzable substrates, p-nitrophenyl-alpha-D-galactopyranoside and 4-methylumbilliferyl-beta-D-glucoside, in the presence of 2.5% sodium deoxycholate. This combination test, performed with a rapid assay for L-pyrrolidonyl-aminopeptidase, could distinguish S. bovis, Streptococcus equinus, Enterococcus spp., Streptococcus pneumoniae, and the viridans group streptococci in culture within 30 min. Twelve species of the genera Streptococcus and Enterococcus were tested. The rapid method correlated well with conventional techniques. The reagents are readily available, inexpensive, and easy to make and can be stored in the refrigerator for at least 6 months.