Au nanoparticle-based probe for selenol in living cells and selenium-rich tea and rice.

Selenocysteine (Sec) is a primary kind of reactive selenium species in cells, and its vital roles in physiological processes have been characterized. Therefore, the highly effective method for sensing Sec in metabolic processes and selenium-rich food must be developed. This study presents a new fluorescent probe, namely, GSH-NB@AuNPs, for highly selective detection of selenol based on the fluorescence quenching quality on the surface of gold nanoparticles (AuNPs). The probe consists of glutathione (GSH) and Nile blue (NB) moieties assembled on AuNPs. The probe exhibits excellent sensitivity and selectivity for Sec and is applied in imaging endogenous and exogenous Sec in living cells through confocal fluorescence microscopy. The proposed probe provides a promising and powerful method for detecting selenol in foodstuff (such as selenium-rich rice and tea) with the detection limit of 9.5 nM.

Lecithin doped electrospun poly(lactic acid)-thermoplastic polyurethane fibers for hepatocyte viability improvement.

The development of hepatocyte cultures in vitro holds great significance in the study of bioartificial liver support systems. Electrospun fiber cultures have received widespread attention as an effective method to culture hepatocytes in vitro. Polylactic acid (PLA) -a synthetic polymer with high biocompatibility and biodegradability- is widely used to fabricate electrospun fibers in the biomedical field. However, the use of PLA is limited in cell cultures due to its brittleness, strong hydrophobicity, and lack of biologically active functional groups. In this study, thermoplastic polyurethane (TPU) and lecithin (Lec) were used to modify PLA by spiking them into the PLA electrospun solution in attempt to establish a suitable fiber scaffold for hepatocyte culture in bioreactors. TPU and lecithin incorporation into PLA increases the flexibility, hydrophilicity, and biologically active groups of the fibers which further promotes the growth, proliferation, and viability of hepatocytes. The morphology, wettability, and biocompatibility of the as-prepared PLA-TPU-Lec fibers were carefully characterized. The results showed that the PLA-TPU-Lec fibers possessed favorable morphology and hydrophilicity, as well as high biocompatibility ability. HepG2 cells on the PLA-TPU-Lec fibers and tissue culture plates (TCP) were exposed to hepatotoxins for 24 h and we found that HepG2 cells on the PLA-TPU-Lec fibers had higher viability than cells on TCP. The PLA-TPU-Lec fibers are therefore expected to be used in vitro for hepatocyte culture to improve cellular activity in artificial liver bioreactors.

Low Cerebral Exposure Cannot Hinder the Neuroprotective Effects of Panax Notoginsenosides.

A bidirectional route of communication between the gastrointestinal tract and the central nervous system, termed the "gut-brain axis," is becoming increasingly relevant to treatment of cerebral damage. Panax Notoginsenoside extract (PNE) is popular for prevention and treatment of cardio-cerebrovascular ischemic diseases although plasma and cerebral exposure levels are extremely low. To date, the mechanisms underlying the neuroprotective effects of PNE remain largely unknown. In the present study, the neuroprotective effects of PNE were systematically studied via investigation of the regulation by PNE of the gastrointestinal microbial community and γ aminobutyric acid (GABA) receptors. The results demonstrated that pretreatment with PNE exerted a remarkable neuroprotective effect on focal cerebral ischemia/reperfusion (I/R) injury in rats, and the efficiency was attenuated in germ-free rats. Pretreatment with PNE could significantly prevent downregulation of Bifidobacterium longum (B.L) caused by I/R surgery, and colonization by B.L could also exert neuroprotective effects. More importantly, both PNE and B.L could upregulate the expression of GABA receptors in the hippocampus of I/R rats, and coadministration of a GABA-B receptor antagonist could significantly attenuate the neuroprotective effects of PNE and B.L. The study above suggests that the neuroprotective effects of PNE may be largely attributable to its regulation of intestinal flora, and oral treatment with B.L was also useful in therapy of ischemia/reperfusion injury (I/R) by upregulating GABA-B receptors.

Qualitatively and quantitatively investigating the regulation of intestinal microbiota on the metabolism of panax notoginseng saponins.

ETHNOPHARMACOLOGICAL RELEVANCE: Intestinal microflora plays crucial roles in modulating pharmacokinetic characteristics and pharmacological actions of active ingredients in traditional Chinese medicines (TCMs). However, the exact impact of altered intestinal microflora affecting the biotransformation of TCMs remains poorly understood. AIMS OF THE STUDY: This study aimed to reveal the specific enterobacteria which dominate the metabolism of panax notoginseng saponins (PNSs) via exploring the relationship between bacterial community structures and the metabolic profiles of PNSs. MATERIALS AND METHODS: 2, 4, 6-Trinitrobenzenesulphonic acid (TNBS)-challenged and pseudo germ-free (pseudo GF) rats, which prepared by treating TNBS and antibiotic cocktail, respectively, were employed to investigate the influence of intestinal microflora on the PNS metabolic profiles. Firstly, the bacterial community structures of the conventional, TNBS-challenged and pseudo GF rat intestinal microflora were compared via 16S rDNA amplicon sequencing technique. Then, the biotransformation of protopanaxadiol-type PNSs (ginsenoside Rb1, Rb2 and Rd), protopanaxatriol-type PNSs (ginsenoside Re, Rf, Rg1 and notoginsenoside R1) and Panax notoginseng extract (PNE) in conventional, TNBS-challenged and pseudo GF rat intestinal microbiota was systematically studied from qualitative and quantitative angles based on LC-triple-TOF/MS system. Besides, glycosidases (ß-glucosidase and ß-xylosidase), predominant enzymes responsible for the deglycosylation of PNSs, were measured by the glycosidases assay kits. RESULTS: Significant differences in the bacterial community structure on phylum, class, order, family, and genera levels were observed among the conventional, TNBS-challenged and pseudo GF rats. Most of the metabolites in TNBS-challenged rat intestinal microflora were identified as the deglycosylation products, and had slightly lower exposure levels than those in the conventional rats. In the pseudo GF group, the peak area of metabolites formed by loss of glucose, xylose and rhamnose was significantly lower than that in the conventional group. Importantly, the exposure levels of the deglycosylated metabolites were found have a high correlation with the alteration of glycosidase activities and proteobacteria population. Several other metabolites, which formed by oxidation, dehydrogenation, demethylation, etc, had higher relative exposure in pseudo GF group, which implicated that the up-regulation of Bacteroidetes could enhance the activities of some redox enzymes in intestinal microbiota. CONCLUSION: The metabolism of PNSs was greatly influenced by intestinal microflora. Proteobacteria may affect the deglycosylated metabolism of PNSs via regulating the activities of glycosidases. Besides, up-regulation of Bacteroidetes was likely to promote the redox metabolism of PNSs via improving the activities of redox metabolic enzymes in intestinal microflora.

Development of a novel sectional multiple filtering scheme for rapid screening and classifying metabolites of ziyuglycoside II in rat liver and excreta specimen based on high-resolution mass spectrometry.

Ziyuglycoside II, one of the major effective ingredients of Sanguisorba officinalis L., had various pharmacological activities including anticancer, anti-inflammation and anti-oxidation, etc. Better understanding of the pharmacology and toxicology of ziyuglycoside II requires the detailed elucidation of its biologic fates in vivo. Herein, the metabolic fate of ziyuglycoside II in rats was investigated based on liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS). To accelerate and simplify the process of metabolite identification from complicated biological matrix, the sectional multiple filtering (SMF) scheme was designed according to the relationship among the molecular weight (MW), mass defect (MD) and retention time (tR) of the metabolites. SMF-I (MW: 700-850Da, MD: 0.40-0.45Da, tR: 4.0-10.0min), SMF-II (MW: 550-700Da, MD: 0.30-0.40Da, tR: 6.0-14.0min) and SMF-III (MW: 400-550Da, MD at 0.25-0.35Da, tR at 9.5-16.0min) were built and utilized to screen phase II conjugations and phase I redox metabolites and deglycosylated derivatives, respectively. As a result, dozens of metabolites, including glucuronic conjugates, hydroxylation, oxidization, dehydration and deglycosylation products, were rapidly discovered, classified and structural identified in rat urine and feces based on SMF scheme and accurate MS(1)/MS(2) information. Obviously, the SMF technique showed superior efficiency and selectivity in ziyuglycoside II metabolite identification. More importantly, SMF would find its extensive application in, but not limited to, the metabolic study for single drug or homologous compounds in traditional Chinese medicine.

Comprehensive characterization of the in vitro and in vivo metabolites of ziyuglycoside I in rat microsome, intestinal flora, excretion specimen and fresh tissues based on LC-Q-TOF/MS.

Ziyuglycoside I is one of the major active ingredients in Sanguisorba officinalis, a popular medicinal plant in China. In the present study, the metabolites of ziyuglycoside I in rat liver microsome and intestinal flora were identified and structurally characterized, and the metabolic rules were summed based on the LC-Q-TOF/MS system. Then, the metabolites in rat excreta samples were rapidly screened and identified according to the in vitro metabolic rules. Finally, ziyuglycoside I was incubated with fresh liver/lung/kidney/stomach homogenates to further explore the source of the metabolites and reveal the possible metabolic organs involved. Four metabolites in liver microsome were identified as M0-Glu, M0-CH2OH, M0-Glu+CH3, M0-Glu-Ara+CH3. In intestinal flora incubation system, 6 degradation products including M0-Glu-Ara+O, M0-Ara, M0-Glu-COOH, M0-Glu, M0-Glu-Ara+O and M0-Ara+H2O were tentatively identified by interpretation of their accurate MS(1) and MS(2) data. Fifteen metabolites in rat urine and feces were identified, and most of the metabolites were attributed to the transformation in liver microsome and intestinal flora. Specifically, more than a dozen of new metabolites were identified in rat fresh tissues, and ziyuglycoside II was confirmed as the major metabolite in rats.

Appropriate choice of collision-induced dissociation energy for qualitative analysis of notoginsenosides based on liquid chromatography hybrid ion trap time-of-flight mass spectrometry.

Liquid chromatography hybrid ion trap/time-of-flight mass spectrometry possessesd both the MS(n) ability of ion trap and the excellent resolution of a time-of-flight, and has been widely used to identify drug metabolites and determine trace multi-components for in natural products. Collision energy, one of the most important factors in acquiring MS(n) information, could be set freely in the range of 10%-400%. Herein, notoginsenosides were chosen as model compounds to build a novel methodology for the collision energy optimization. Firstly, the fragmental patterns of the representatives for the authentic standards of protopanaxadiol-type and protopanaxatriol-type notoginsenosides authentic standards were obtained based on accurate MS(2) and MS(3) measurements via liquid chromatography hybrid ion trap/time-of-flight mass spectrometry. Then the extracted ion chromatograms of characteristic product ions of notoginsenosides in Panax Notoginseng Extract, which were produced under a series of collision energies and, were compared to screen out the optimum collision energies values for MS(2) and MS(3). The results demonstrated that the qualitative capability of liquid chromatography hybrid ion trap/time-of-flight mass spectrometry was greatly influenced by collision energies, and 50% of MS(2) collision energy was found to produce the highest collision-induced dissociation efficiency for notoginsenosides. BesidesAddtionally, the highest collision-induced dissociation efficiency appeared when the collision energy was set at 75% in the MS(3) stage.

Development and validation of an UFLC-MS/MS assay for the absolute quantitation of nine notoginsenosides in rat plasma: Application to the pharmacokinetic study of Panax Notoginseng Extract.

Notoginsenosides, the main active gradients of Chinese traditional medicine Panax notoginseng, possesses a variety of biological activities including antioxidant property, anti-hyperglycemic, anti-obese, etc. However, pharmacokinetic evaluation for notoginsenosides is still a formidable task due to their low concentrations and complex components in vivo. The summation of this work generated a rapid and sensitive method for quantitative analysis of multi-notoginsenoside in rat plasma based on ultra fast liquid chromatographic-tandem mass spectrometric. After liquid-liquid extraction by n-butanol, notoginsenoside R1, Rg3, Rd, Rg2, Rb2, Rf, Rg1, Rb1 and Re were simultaneously monitored in negative ionization mode after separating on a Thermo ODS C18 column (5mm 50mm×2.1mm) by a binary gradient elution, and all compounds were analyzed within 9min. Multiple reaction monitoring (MRM) was performed as follows: R1 (m/z 967.7→637.4), Rg3 (m/z 819.6→621.4), Rd (m/z 981.6→783.5), Rg2 (m/z 819.6→475.4), Rb2 (m/z 1113.4→783.4), Rf (m/z 835.6→475.4), Rg1 (m/z 835.6→637.6), Rb1 (m/z 1143.7→945.6), Re (m/z 981.6→637.4), internal standard (digoxin, m/z 815.5→779.4). Validation parameters (linearity, sensitivity, intra-and inter-assay precision and accuracy, recovery and matrix effect) were within acceptable ranges and biological extracts were stable during the entire storing and preparing process. This UFLC-MS/MS approach was further validated by being applied to the pharmacokinetic study for P. Notoginseng extract in rats, and the pharmacokinetic parameters were calculated by Winolin software. Thus, the presently developed methodology was simple, robust, accurate, precise, and would be useful for the pharmacokinetic studies for all kinds of notoginsenosides and other herbal saponins.