A deep-red fluorescent molecular rotor based on donor-two-acceptor modular system for imaging mitochondrial viscosity.

A new donor-two-acceptor modular fluorescence rotor DpCy7 involving a phenolate donor unit and two benzothiazolium acceptor moieties was designed and synthesized. The DpCy7 underwent an internal charge transfer to form a Cy7-like longer conjugated system fluorochrome at a physiological pH. The probe exhibited a strong turn-on (8.5-fold) deep-red emission with a larger Stokes shift in glycerol aqueous solutions with restriction of rotation. Both the fluorescence intensity and fluorescence lifetime displayed the linear relationship of viscosity changes in the logarithmic plots. Furthermore, the HeLa cell imaging experiments of DpCy7 indicated that the rotor could be used to monitor the mitochondrial viscosity in living cells. This new type of deep-red fluorescence rotor provides a potential platform for determining viscosity at subcellular levels.

Comparative analysis of constitutes and metabolites for traditional Chinese medicine using IDA and SWATH data acquisition modes on LC-Q-TOF MS.

Identification of components and metabolites of traditional Chinese medicines (TCMs) employing liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF MS) techniques with information-dependent acquisition (IDA) approaches is increasingly frequent. A current drawback of IDA-MS is that the complexity of a sample might prevent important compounds from being triggered in IDA settings. Sequential window acquisition of all theoretical fragment-ion spectra (SWATH) is a data-independent acquisition (DIA) method where the instrument deterministically fragments all precursor ions within the predefined m/z range in a systematic and unbiased fashion. Herein, the superiority of SWATH on the detection of TCMs' components was firstly investigated by comparing the detection efficiency of SWATH-MS and IDA-MS data acquisition modes, and sanguisorbin extract was used as a mode TCM. After optimizing the setting parameters of SWATH, rolling collision energy (CE) and variable Q1 isolation windows were found to be more efficient for sanguisorbin identification than the fixed CE and fixed Q1 isolation window. More importantly, the qualitative efficiency of SWATH-MS on sanguisorbins was found significantly higher than that of IDA-MS data acquisition. In IDA mode, 18 kinds of sanguisorbins were detected in sanguisorbin extract. A total of 47 sanguisorbins were detected when SWATH-MS was used under rolling CE and flexible Q1 isolation window modes. Besides, 26 metabolites of sanguisorbins were identified in rat plasma, and their metabolic pathways could be deduced as decarbonylation, oxidization, reduction, methylation, and glucuronidation according to their fragmental ions acquired in SWATH-MS mode. Thus, SWATH-MS data acquisition could provide more comprehensive information for the component and metabolite identification for TCMs than IDA-MS.

Systematically identifying the hepatoprotective ingredients of schisandra lignan extract from pharmacokinetic and pharmacodynamic perspectives.

BACKGROUND: Herbal medicines (HMs) have been proven to be productive sources of leads for the development of drugs. To date approximately 150 lignans have been identified from Schisandra sphenanthera. Hepatoprotective activity is a well-known characteristic of schisandra lignans, yet the authentic types of active lignans are still not well known. PURPOSE: The present study aimed to develop a reliable and efficient strategy for identifying the hepatoprotective ingredients of schisandra lignan extract (SLE). METHODS: SLEs were prepared by extracting Schisandra sphenanthera powder using 10%, 50% and 90% ethanol (w/w 1:10) combining 5-fold volume of ethyl acetate. The schisandra lignans in SLEs were qualitatively analyzed based on liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LCMS-IT-TOF). Preparative liquid chromatography (PLC) was used to collect ingredient fractions. The hepatoprotective activity of schisandra lignans was systematically investigated on in vivo and in vitro models. RESULTS: The SLE extracted by 50% ethanol and 5-fold volume of ethyl acetate (50%SLE) had the highest lignan content and exhibited significantly stronger hepatoprotective activity than other SLEs (P <  0.01). The hepatoprotective effect of 50%SLE mainly attributed to the SLE segment which collected from 12 to 22 min by PLC. Schisantherin A (Sth A) was confirmed as the most promising hepatoprotective drug in Schisandra sphenanthera due to high content in crude materials, high exposure level in vivo and high efficiency on APAP-induced hepatotoxicity. CONCLUSION: The hepatoprotective ingredients of SLEs were systematically investigated based on the presently developed approach, and Sth A was identified as the optimum hepatoprotective candidate in Schisandra sphenanthera.

Is the combinational administration of doxorubicin and glutathione a reasonable proposal?

The combinational administration of antioxidants and chemotherapeutic agents during conventional cancer treatment is among one of the most controversial areas in oncology. Although the data on the combinational usage of doxorubicin (DOX) and glutathione (GSH) agents have been explored for over 20 years, the duration, administration route, and authentic rationality have not yet been fully understood yet. In the current study, we systematically investigated the pharmacokinetics (PK) and pharmacodynamics (PD) with both in vivo and in vitro models to elucidate the influence of GSH on the toxicity and efficacy of DOX. We first studied the cardioprotective and hepatoprotective effects of GSH in Balb/c mice, H9c2, and HL7702 cells. We showed that coadministration of exogenous GSH (5, 50, and 500 mg/kg per day, intragastric) significantly attenuated DOX-induced cardiotoxicity and hepatotoxicity by increasing intracellular GSH levels, whereas the elevated GSH concentrations did not affect the exposure of DOX in mouse heart and liver. From PK and PD perspectives, then the influences of GSH on the chemotherapeutic efficacy of DOX were investigated in xenografted nude mice and cancer cell models, including MCF-7, HepG2, and Caco-2 cells, which revealed that administration of exogenous GSH dose-dependently attenuated the anticancer efficacy of DOX in vivo and in vitro, although the elevated GSH levels neither influenced the concentration of DOX in tumors in vivo, nor the uptake of DOX in MCF-7 tumor cells in vitro. Based on the results we suggest that the combined administration of GSH and DOX should be contraindicated during chemotherapy unless DOX has caused serious hepatotoxicity and cardiotoxicity.

Activated charcoal significantly improved the reliability of methods for quantitative analysis of endogenous substances in biological specimens: Glutathione and cysteine as cases.

When developing a quantitative assay for exogenous or endogenous compounds, guidelines for method validation normally recommend that the biological specimens should be prepared in corresponding authentic matrices, yet "analyte-free authentic matrices" is in general not available. It is generally known that GSH and CYS are endogenous compounds and present in both prokaryotes and eukaryotes. Herein, an efficient approach for the quantitative analysis of endogenous substances in biological specimens was developed, and glutathione (GSH) & cysteine (CYS) were chosen as model endogenous substances. Activated carbon (AC), a common adsorbent for the adsorption of environmental pollutants, was used to remove the endogenous GSH and CYS and prepare "GSH&CYS-free biological matrix". The endogenous GSH and CYS in mouse plasma, blood and liver homogenate were found can be almost removed via incubating with 100 mg of AC for 2 h. After optimizing the derivatization reagents, internal standard and analytical parameters, a reliable quantitative assay of GSH and CYS in mouse plasma, blood and liver homogenate was developed and validated on LC-ESI-MS/MS using corresponding AC-adsorbed mouse biological matrices. The validation results indicated that the developed method provided suitable accuracy, sensitivity, specificity and high throughput for the analysis of GSH and CYS. Finally, the developed LC-ESI-MS/MS assay was successfully applied to measure the concentrations of GSH and CYS in liver injury mice. The presently developed methodology could be widely applied in the quantitative analysis of endogenous compounds in various complex mixtures such as biological, herbal and environmental samples.

Pharmacokinetic and pharmacodynamic evidence for developing an oral formulation of octreotide against gastric mucosal injury.

Among the somatostatin analogues, octreotide (OCT) is the most commonly used in clinic via intravenous or subcutaneous injection to treat various diseases caused by increased secretion of growth hormone, gastrin or insulin. In order to assesse the feasibility of developing oral formulations of OCT, we conducted systematical pharmacokinetic and pharmacodynamic analyses of OCT in several animal models. The pharmacokinetic studies in rats showed that intragastric administration of OCT had extremely low bioavailability (<0.5%), but it could specifically distribute to the gastric mucosa due to the high expression of somatostatin receptor 2 (SSTR2) in the rat stomach. The pharmacodynamic studies revealed that intragastric administration of OCT dose-dependently protected against gastric mucosal injury (GMI) in mice with WIRS-induced mouse gastric ulcers, which were comparable to those achieved by intravenous injection of OCT, and this effect was markedly attenuated by co-administration of CYN-154806, an antagonist of SSTR2. In pyloric ligation-induced ulcer mice, we further demonstrated that OCT significantly reduced the secretion of gastric acid via down-regulating the level of gastrin, which was responsible for the protective effect of OCT against GMI. Overall, we have provided pharmacokinetic and pharmacodynamic evidence for the feasibility of developing an oral formulation of OCT. Most importantly, the influence of SSTR2 on the pharmacokinetics and pharmacodynamics of OCT suggested that an oral formulation of OCT might be applicable for other clinical indications, including neuroendocrine neoplasms and pituitary adenoma due to the overexpression of SSTR2 on these tumor cells.

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.

An integrated strategy for the quantitative analysis of endogenous proteins: A case of gender-dependent expression of P450 enzymes in rat liver microsome.

Liquid chromatography mass spectrometry based methods provide powerful tools for protein analysis. Cytochromes P450 (CYPs), the most important drug metabolic enzymes, always exhibit sex-dependent expression patterns and metabolic activities. To date, analysis of CYPs based on mass spectrometry is still facing critical technical challenges due to the complexity and diversity of CYP isoforms besides lack of corresponding standards. The aim of present work consisted in developing a label-free qualitative and quantitative strategy for endogenous proteins, and then applying to the gender-difference study for CYPs in rat liver microsomes (RLMs). Initially, trypsin digested RLM specimens were analyzed by the nanoLC-LTQ-Orbitrap MS/MS. Skyline, an open source and freely available software for targeted proteomics research, was then used to screen the main CYP isoforms in RLMs under a series of criteria automatically, and a total of 40 and 39 CYP isoforms were identified in male and female RLMs, respectively. More importantly, a robust quantitative method in a tandem mass spectrometry-multiple reaction mode (MS/MS-MRM) was built and optimized under the help of Skyline, and successfully applied into the CYP gender difference study in RLMs. In this process, a simple and accurate approach named 'Standard Curve Slope" (SCS) was established based on the difference of standard curve slopes of CYPs between female and male RLMs in order to assess the gender difference of CYPs in RLMs. This presently developed methodology and approach could be widely used in the protein regulation study during drug pharmacological mechanism research.

Optimization and evaluation of MALDI TOF mass spectrometric imaging for quantification of orally dosed octreotide in mouse tissues.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF-MSI) has received considerable attention in recent years since it allows molecular mapping of diverse bimolecular in animal/plant tissue sections, although some barriers still exist in absolute pixel-to-pixel quantification. Octreotide, a synthetic somatostatin analogue, has been widely used to prevent gastrointestine bleeding in the clinic. The aim of the present study is to develop a MALDI-TOF-MSI method for quantitatively visualizing spatial distribution of octreotide in mouse tissues. In this process, a structurally similar internal standard was spotted onto tissue section together with matrix solution to minimize signal variation and give excellent quantitative results. The 2,5-dihydroxybenzoic acid was chosen as the most suitable matrix via comparing the signal/noise generated by MALDI-TOF-MSI after cocrystallization of octreotide with different matrix candidates. The reliability of MALDI-TOF-MSI, with respect to linearity, sensitivity and precision, was tested via measuring octreotide in fresh tissue slices at different concentrations. The validated method was then successfully applied to visualize the distribution of octreotide in mouse tissues after oral administration of octreotide at 20mg/kg. The results demonstrated that MALDI-TOF-MSI could not only clearly visualize the spatial distribution of octreotide, but also make the calculation of the key pharmacokinetic parameters (Tmax and t1/2) possible. More importantly, the tissue concentration-time curves of octreotide determined by MALDI-TOF-MSI agreed well with those measured based on LC-MS/MS.These findings illustrate the potential of MALDI-TOF-MSI in pharmacokinetic profiling during drug development.

Bioanalytical assay development and validation for simultaneous quantification of five schisandra lignans in rat primary hepatocytes based on LC-MS/MS: application to a real-time uptake study for Schisandra Lignan Extract.

Schisandra lignans, mainly including schizandrol A, schizandrol B, schisantherin A, schizandrin A, schizandrin B, etc., are the major active ingredients of Schisandra chinensis. In the present study, a robust liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the simultaneous quantification of schisandra lignans in rat primary hepatocytes. Lovastatin was used as an internal standard, and chromatographic separation was achieved on a Shimadzu C18 column with a gradient elution at the flow rate of 0.2 mL/min. All of the analytes were detected in multiple reaction monitoring mode with positive electrospray ionization since the sodium adduct ion [M + Na]+ was observed as the most intensive peak in the MS spectrum. For schizandrol A, schisantherin A and schizandrin A, the dynamic range was within 2-1000 ng/mg protein, and the linear range of schizandrol B and schizandrin B was from 5 to 1000 ng/mg protein. The intra- and inter-day precision was <15% and the accuracy (relative error) ranged from -15 to 15%. No significant variation was observed in the stability tests. The validated method was then successfully applied to the time-dependent uptake study for the Schisandra Lignan Extract in rat primary hepatocytes.

Pharmacokinetic study based on a matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight imaging mass microscope combined with a novel relative exposure approach: A case of octreotide in mouse target tissues.

Application of imaging mass spectrometry in drug pharmacokinetics remains challenging due to its weak quantitative capability. Herein, an imaging mass microscope (iMScope), equipped with an optical microscope, an atmospheric pressure ion-source chamber for matrix-assisted laser desorption/ionization (AP-MALDI) and a hybrid quadrupole ion trap time-of-flight (QIT-TOF) analyzer, was first validated and applied to visualize drug disposition in vivo. The distribution and elimination rate of the therapeutic peptide octreotide, a long-acting analogue of the natural hormone somatostatin, was first calculated based on the data determined by iMScope system combining a novel relative exposure strategy. Microspotted pixel-to-pixel quantitative iMScope provided a relative amount of octreotide presented in a thin stomach/intestinal section while preserving its original spatial distribution. The images of dosed mouse stomach clearly demonstrated the transport process of octreotide from the mucosal layer to the muscle side. More importantly, octreotide was found to eliminate from the intestines rapidly, the absorption peak time (Tmax) appeared at 40 min and the half-life time (t1/2) was calculated as 37.7 min according to the elimination curves. Comparisons to the LC-MS/MS data adequately indicated that the quantification approach and methodology based on the iMScope was reliable and practicable for drug pharmacokinetic study.

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.

The metabolic and pharmacokinetic studies for HM-3 in rats based on LC-Q-TOF/MS and LC-MS/MS combing a convenient biological sample processing method.

In this contribution, the metabolic and pharmacokinetic characteristics for the therapeutic peptide HM-3 were investigated using LC-Q-TOF/MS and LC-MS/MS systems combing a fast biological sample processing method. According to the accurate MS(1) and MS(2) data generated by LC-Q-TOF/MS, a total of 6 metabolites in rats were detected and tentatively identified as the degradation products which formed by successive loss of amino acid from HM-3. The structures of the 2 main metabolites (M1 and M2) were confirmed by comparing the chromatographic and mass spectrographic characteristics with the corresponding synthetic standards. Then, an absolute quantitative analysis method based on LC-MS/MS system was built and fully validated with respect to linearity, sensitivity, accuracy, precision, matrix effect, stability, etc. The results indicated that HM-3, M1 and M2 were linear in peak area ratios over the concentration range of 0.5-200.0ng/mL with a correlation coefficient>0.99. The intra-day and inter-day precisions (RSD%) were less than 15%, and the accuracy was below 10% in terms of RE%. The validated method was then successfully applied to the studies of preclinical pharmacokinetics for HM-3.

[Transient Storage Characteristics of Artificial Pool Geomorphic Structure in an Agricultural Headwater Stream].

From November 2015 to February 2016, five short-term tracer injections were performed with a conservative tracer (NaCl) in an agricultural headwater stream, Lake Chaohu basin. Thus the data sets of tracer experiments were finally employed for calculating the physical characteristics and transient storage metrics. Through the comparisons between the artificial pool reach and straight reach, characteristics of transient storage for the artificial pool geomorphic structure were interpreted and explored. Study results showed that: ① The ratio of As/A in artificial pool was larger than that in straight reach, whereas its value of exchange coefficient α was lower by an order of magnitude than that of straight reach. ② Artificial pool geomorphic structure had greater influence of transient storage on solute retention than that in straight reach, but its solute retention capacity of flowing water was weaker than that of straight reach. ③ It had a large ratio of As/A for the pool geomorphic structure, whereas its impact on the migration and transformation of solutes was less than that of straight reach. ④ Based on the Fmed200 metric, the transient storage accounted for 18.86% to 26.05% of travel time in artificial pool. For the straight reach, the Fmed200 metric had a range of 5.28% to 33.87%. In most cases, the values of Fmed200 metric in straight reach were higher than those in artificial pool. ⑤ Significant differences existed between artificial pool geomorphic structure and straight reach in the values of φw, φA and Ts, however, the differences were not significant in other indicators.

Monitoring of flow field based on stable isotope geochemical characteristics in deep groundwater.

The water circulation in deep aquifers controls not only chemical composition of the groundwater, but also stable isotope composition. In order to analyze the flow field in the process of the deep groundwater circulation in different aquifers, specimens belonging to the fourth aquifer in the Quaternary (the fourth aquifer for short), the coal and sandstone cranny aquifer in the Permian, and Carboniferous (the coal catena aquifer for short), the Taiyuan group limestone aquifer in the Carboniferous (the Taiyuan limestone aquifer for short), and the limestone aquifer in the Ordovician (the Ordovician limestone aquifer for short) were gained from the top down in Renlou colliery and local Linhuan coalmine district, northern Anhui, China, in the study. δD, δ(18)O, and the content of tall dissolve solids (TDS for short) of these specimens were tested. The experimental results had revealed that the groundwater in the fourth aquifer and the Taiyuan limestone aquifer takes on (18)O excursion and the coal catena aquifer takes on D excursion in Linhuan coalmine district, while excursion characteristic in the Ordovician limestone aquifer is not evident in the coalmine district. By analysis, δ(18)O and the content of TDS are in negative relationship in the groundwater of the fourth aquifer and the Taiyuan limestone aquifer in Linhuan coalmine district, yet δD and the content of TDS are in positive relationship in the coal catena aquifer. Mining greatly influences the fourth aquifer and the coal catena aquifer so the groundwater in the fourth aquifer flows from northwest and southeast to mining areas and the groundwater in the coal catena aquifer flows from around to mining areas. However, mining does not influence the Taiyuan limestone aquifer evidently so the groundwater flows from east to west still.