Liquid chromatography/quadrupole time-of-flight mass spectrometry in combination with online hydrogen/deuterium exchange technique for structural elucidation of phase I metabolites of iso-phenylcyclopentylamine in rat bile.

MS/MS experiment and accurate mass measurement are powerful tools in metabolite identification. However, sometimes these data do not provide enough information to assign an unambiguous structure to a metabolite. In combination with MS techniques, hydrogen/deuterium (H/D) exchange can provide additional information for structural elucidation by determination of the number of exchangeable hydrogen atoms in a structure. In this study, the principal phase I metabolites of iso-phenylcyclopentylamine in rat bile were identified by high-performance liquid chromatography with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS). Since N-oxidation may occur because of the existence of the primary amino group in the structure, it was difficult to differentiate the hydroxylated metabolites from N-oxides by ESI-Q-TOF-MS alone. Therefore, online H/D exchange technique was applied to solve this problem. Finally, 25 phase I metabolites were detected and structurally described, in which 11 were confirmed to be N-oxides. This study demonstrated the effectiveness of high-resolution mass spectrometry in combination with an online H/D exchange technique in rapid identification of drug metabolites, especially in discriminating hydroxylated metabolites from N-oxides.

Inclusion complex of quercetin with sulfobutylether ß-cyclodextrin: preparation, characterization, antioxidant and antibacterial activities and the inclusion mechanism.

Quercetin (QCT) has a variety of pharmacological effects, such as antioxidant, antibacterial, anticancer, anticardiovascular and antiaging effects. However, its poor water solubility, stability and bioavailability limit its applications. The special structure of cyclodextrins and their derivatives with a hydrophobic inner cavity and hydrophilic outer wall can load a variety of hydrophobic drugs of a suitable size and shape, thereby improving the stability and solubility of these molecules. In this study, an inclusion complex of quercetin and sulfobutylether-ß-cyclodextrin was prepared. It was characterized via FT-IR, UV, 1H NMR, XRD, DSC, and SEM analysis, which revealed the successful formation of the inclusion complex. In vitro biological activity estimations were carried out and the results indicated that the inclusion complex displayed higher antioxidative and antibacterial properties compared with free QCT. In addition, the mechanisms of inclusion were explored using 1H NMR analysis and docking calculations, thus providing a theoretical basis for obtaining an inclusion complex.

Polysaccharide NAP-3 Synergistically Enhances the Efficiency of Metformin in Type 2 Diabetes via Bile Acid/GLP-1 Axis through Gut Microbiota Remodeling.

The polysaccharides of edible mushrooms are excellent phytochemicals for adjuvant treatment of metabolic diseases, but the potential mechanisms of synergistic effects are unclear. In this work, we discovered that NAP-3 enhanced the efficiency of metformin in lipid and glucose metabolism in type 2 diabetic (T2D) mice in a gut microbiome-dependent way. NAP-3 remodeled the intestinal microbial, resulting in the decreased activity of bile salt hydrolases and upregulation of CYP27A1 and CYP7B1 functions in the alternative pathway of bile acid synthesis, which leads to accumulation of the conjugated bile acids in ileum, specifically TßMCA and TUDCA. The accumulated conjugated bile acids either blocked or stimulated the nuclear receptors Farnesoid-X-receptor and TGR5, inducing the release of GLP-1 and ultimately enhanced glucose metabolism in mice. Collectively, our research indicated that edible mushroom polysaccharide NAP-3 may serve as a promising adjunctive oral therapeutic agent for T2D.

Analysis of the traditional medicine YiGan San by the fragmentation patterns of cadambine indole alkaloids using HPLC coupled with high-resolution MS.

YiGan San (YGS) has long been used in traditional Japanese and Chinese folk medicine and serves as a potent and novel therapeutic agent to treat Alzheimer's disease. In the present study, a rapid and sensitive method based on HPLC coupled with diode-array detection and quadrupole TOF MS (Q-TOF-MS) was designed to reveal the chemical constituents of YGS. Thirty-six compounds were identified and assigned in YGS, including 14 alkaloids, nine γ-lactones, six flavonoids, three triterpenoid saponinares, two small molecular organic acids, and two other types of compounds. In addition, the accurate fragment weight and MS/MS fragmentation reactions of a subtype indole alkaloid in Uncariae ramulus cum uncis were summarized for the first time to realize rapid identification without reference substances. For the first time, 11 major constituents were comprehensively quantified with a HPLC coupled with triple-quadrupole MS method. A three-section switch was used to realize such multicomponent identification. The contents of saikosaponin B2 and isoliquiritin, which produce anti-inflammatory and antidepressant-like effects, were extremely different, up to 700 times, in two sources of YGS. The developed qualitative and quantitative method was proved to be precise, accurate, and reproducible.

Development and validation of a simple, sensitive and accurate LC-MS/MS method for the determination of guanfacine, a selective α2A -adrenergicreceptor agonist, in plasma and its application to a pharmacokinetic study.

A simple, practical, accurate and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for the quantitation of guanfacine in beagle dog plasma. After protein precipitation by acetonitrile, the analytes were separated on a C18 chromatographic column by methanol and water containing 0.1% (v/v) formic acid with a gradient elution. The subsequent detection utilized a mass spectrometry under positive ion mode with multiple reaction monitoring of guanfacine and enalaprilat (internal standard) at m/z 246.2 → 159.0 and m/z 349.2 → 205.9, respectively. Good linearity was obtained over the concentration range of 0.1-20 ng/mL for guanfacine in dog plasma and the lower limit of quantification of this method was 0.1 ng/mL. The intra- and inter-day precisions were <10.8% relative standard deviation with an accuracy of 92.9-108.4%. The matrix effects ranged from 89.4 to 100.7% and extraction recoveries were >90%. Stability studies showed that both analytes were stable during sample preparation and analysis. The established method was successfully applied to an in vivo pharmacokinetic study in beagle dogs after a single oral dose of 4 mg guanfacine extended-release tablets.

Triterpenes from Poria cocos are revealed as potential retinoid X receptor selective agonists based on cell and in silico evidence.

Poria cocos is an edible and medicinal fungus that is widely used in Traditional Chinese Medicines as well as in modern applications. Retinoid X receptor (RXR) occupies a central place in nuclear receptor signaling, and a pharmacological RXR-dependent pathway is involved in myeloid cell function. Here, structural information for 82 triterpenes from P. cocos and 17 known RXR agonists was collected in a compound library and retrieved for a molecular docking study. Three triterpenes, 16α-hydroxytrametenolic acid (HTA), pachymic acid (PA), and polyporenic acid C (PPAC), were identified as novel RXR-specific agonists based on luciferase reporter assays and in silico evidence. Treatment with HTA, PA, and PPAC significantly induced differentiation of the human promyelocytic leukemia cell line HL-60 with EC50 values of 21.0 ± 0.52, 6.7 ± 0.37, and 9.4 ± 0.65 µM, respectively. These effects were partly blocked by the RXR antagonist UVI3003, suggesting that an RXR-dependent pathway may play an important role in their anti-acute promyelocytic leukemia (APL) effects. Taken together, triterpenes from P. cocos are revealed as naturally occurring RXR selective agonists with the potential for anti-cancer activity. These results suggest a novel approach to the treatment or prevention of APL.

Inclusion Complexes of Lycopene and ß-Cyclodextrin: Preparation, Characterization, Stability and Antioxidant Activity.

In this study, the inclusion complexes of lycopene with ß-cyclodextrin (ß-CD) were prepared by the precipitation method. Then the inclusion complexes were characterized by the scanning electron microscopy (SEM), ultraviolet-visible spectroscopy (UV), microscopic observation, liquid chromatography, differential scanning calorimetry (DSC) and phase-solubility study. Moreover, the stability and antioxidant activity were tested. The results showed that lycopene was embedded into the cavity of ß-CD with a 1:1 stoichiometry. Moreover, the thermal and irradiant stabilities of lycopene were all significantly increased by the formation of lycopene/ß-CD inclusion complexes. Antioxidant properties of lycopene and its inclusion complexes were evaluated on the basis of measuring the scavenging activity for 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl and superoxide anion radicals. The results showed that the scavenging activity of DPPH radicals was obviously increased by the formation of the inclusion complex with ß-cyclodextrin at concentrations of 5-30 µg/mL, however, some significant positive effects on the scavenging activity of hydroxyl and superoxide anion radicals were not observed and the reasons are worth further study.

A new lysosome-targetable fluorescent probe with a large Stokes shift for detection of endogenous hydrogen polysulfides in living cells.

Hydrogen polysulfides (H2Sn, n > 1) influence a variety of important biological functions and activities associated with hydrogen sulfide (H2S). The development of probes for rapid, selective, and sensitive detection of H2Sn still remains a great challenge. Lysosome plays crucial roles in various physiological processes among living cells, which arouse high interest in detecting endogenous lysosome-targetable H2Sn. To the best of our knowledge, there is no lysosome-targetable probe for H2Sn has been reported. In this work, a new lysosome-targetable probe NIPYNF, based on the scaffold of imidazo [1,5-]pyridine, with a large Stokes shift (215 nm), low detection limit (84 nM), fast response time (6 min) and low cytotoxicity was designed and synthesized. Furthermore, NIPYNF was successfully applied into the cell imaging for detection of endogenous lysosome-targetable H2Sn.

Wound Healing Activity of a Skin Substitute from Residues of Culinary-Medicinal Winter Mushroom Flammulina velutipes (Agaricomycetes) Cultivation.

A skin substitute TG05 obtained from residues of the culinary-medicinal mushroom Flammulina velutipes cultivation process was developed in this study for the first time. Pre-column derivatization high-performance liquid chromatography fingerprints analysis revealed that TG05 was composed of water-insoluble fibers containing xylose (57%), glucose (19.5%), and arabinose (16.3%) as major monomers. Porous and opaque structure of TG05 was demonstrated by scanning electron microscopy. Animal experiments conducted on mice and rats indicated that TG05 notably accelerated the wound-healing process. In addition, TG05 induced proliferation and migration of human keratinocytes time- and dose-dependently. Taken together, the skin substitute TG05 with new structure promotes wound healing in vitro and in vivo. This study provided a novel method to produce functional biomaterial from abundant and low-cost agricultural residues generated during edible mushroom cultivation.

16α-Hydroxytrametenolic Acid from Poria cocos Improves Intestinal Barrier Function Through the Glucocorticoid Receptor-Mediated PI3K/Akt/NF-κB Pathway.

This study evaluated the effect of triterpenoids from edible mushroom Poria cocos on intestinal epithelium integrity and revealed the transcriptional regulatory pathways that underpin restorative mechanisms in the gut. Based on computational docking studies, transcriptional activation experiments and glucocorticoid receptor (GR) protein immunofluorescence localization assays in cultured cells, 16α-hydroxytrametenolic acid (HTA) was discovered as a novel GR agonist in this study. HTA ameliorates TNF-α-induced Caco-2 monolayer intestinal epithelial barrier damage and suppressed activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt), which attenuated downstream IκB and nuclear factor kappa-B (NF-κB) phosphorylation through GR activation. Moreover, HTA prevented NF-κB translocation into the nucleus and binding to its cis-element and suppressed lipopolysaccharide-induced downstream NO production and pro-inflammatory cytokines at both protein and mRNA expression levels. In conclusion, HTA from P. cocos improves intestinal barrier function through a GR-mediated PI3K/Akt/NF-κB signaling pathway and may be potentially exploited as a supportive dietary therapeutic strategy for restoring gut health.

Effects of high hydrostatic pressure on the growth and beta-carotene production of Rhodotorula glutinis.

The effects of high hydrostatic pressure (HHP) on the biomass and beta-carotene biosynthesis of Rhodotorula glutinis R68 were studied. After treatment with five repeated cycles at 300 MPa for 15 min, the barotolerant mutant PR68 was obtained. After 72 h of culture, the biomass of mutant PR68 was 21.6 g/l, decreased by 8.5% compared to the parental strain R68, but its beta-carotene production reached 19.4 mg/l, increased by 52.8% compared to the parental strain R68. The result of restriction fragment length polymorphism (RFLP) analysis suggested that mutant strain PR68 was likely to change in nucleic acid level, and thus enhanced beta-carotene production in this strain as a result of gene mutation induced by HHP treatment.

Simultaneous determination of galactose, glucose, lactose and galactooligosaccharides in galactooligosaccharides raw materials by high-performance anion-exchange chromatography with pulsed amperometric detection.

This study describes a method for the simultaneous determination of galactose, glucose, lactose and galactooligosaccharides (GOS) in GOS raw materials (GOS syrups and powdered GOS) by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The GOS raw materials were extracted with phosphate buffer. The extract was then treated with ß-galactosidase to hydrolyze GOS and lactose. The total amounts of galactose and glucose released from GOS and lactose were determined in the treated solution. Free galactose, glucose and lactose were determined in the initial solution. The glucose in a ß-galactosidase solution was also determined. The content of GOS in GOS raw materials was calculated by the increment of galactose and glucose after GOS were hydrolyzed, and the glucose and galactose also released from lactose were taken into consideration. The validated method has been successfully applied to determine the content of galactose, glucose, lactose and GOS in GOS syrups and powdered GOS.

[Mutation effect of ultra high pressure on microbe].

(Ultra) high pressure had many influences on microbe. It could regulate the expression of gene and protein, influence DNA's structure and function as well as change cell morphology and cell component. These effects not only make (ultra) high pressure to be applied into food sterilization, conserving and some processing, but also indicate it would play an important role in mutagenic breeding of microbe. Pressure can change the structure and function of microbe, yet it is possible that (ultra) high pressure could induce mutation of microbe. Now the feasibility of (ultra) high pressure's mutation effect was discussed according to the effects of it on microbe, some examples and author's studying.

[Determination of congo red in beef by high performance liquid chromatography-tandem quadrupole time of flight mass spectrometry and ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry].

A method was developed for the determination of congo red in beef. The analyte was identified by high performance liquid chromatography-tandem quadrupole time of flight mass spectrometry (LC-QTOF MS) and quantitatively determined by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry. After purified by liquid-liquid extraction, the congo red in the beef sample was separated on an Agilent ZORBAX Eclipse Plus C18 Rapid Resolution HD UPLC column (50 mm x 2.1 mm, 1.8 microm) HPLC , using 95% (volume percentage) methanol as the mobile phase at 0.2 mL/min. The detection was performed on an AB 4000 + triple quadrupole mass spectrometer utilizing electrospray ionization (ESI) interface operated in negative ion mode and multiple-reaction monitoring (MRM) mode. The results showed that the linear range of congo red mass concentration was 0.03 - 1 mg/L with the correlation coefficient of 0.999 8. The method had a good precision with the RSDs lower than 5% and the recoveries ranging from 88% to 91%. The limit of detection (LOD) of congo red was 0.01 mg/L. With good reproducibility, the method is simple, fast and effective for the determination of the illegally added congo red in beef and other meat products.