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A blood-brain barrier microfluidic chip platform for studying the permeability of active components in traditional Chinese medicine was developed. This model used primary human brain microvascular endothelial cells on a microfluidic chip consisting of two perpendicularly-crossing channels and a single layer porous polycarbonate membrane. The physiological shear stress in the human vasculature was also modeled in this device. Cell viability on the chip was monitored by cell staining and immunofluorescence staining. The cells spread well and the structure of an intercellular adhesion protein was satisfactory. The permeability of fluorescent tracers and three model drugs and the functional expression of P-glycoprotein (P-gp)on the blood-brain barrier were investigated. The results show that the apparent permeability coefficients (Papp) of the fluorescent tracers and three model drugs were consistent with those reported in the literature, and P-gp on the chip showed normal function, indicating that there was a complete structure and a functional BBB. The permeability of six active components of traditional Chinese medicine was investigated through this microfluidic chip and the drug concentration was determined by HPLC-MS/MS to obtain the Papp of each component. The Papp of corydaline was (4.51 ± 1.90)×10-7 cm·s-1, the Papp of tetrahydropalmatine was (9.10 ± 6.59)×10-7 cm·s-1, and the Papp of imperatorin was (9.38 ± 2.53)×10-7 cm·s-1; the concentration of isoimperatorin, baicalin and chlorogenic acid was below the limit of quantification, which suggested that isoimperatorin, baicalin and chlorogenic acid have poor permeability in this BBB chip. This blood-brain barrier microfluidic platform possesses a complete barrier function and near-physiological conditions and could be a valuable in vitro tool for drug permeability evaluation.
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Membrane proteins are the main undertakers of biofilm function, and also the most important target group for innovative drug discovery and research. About 60% of drugs targets are membrane proteins. Due to the obvious aggregation and denaturation tendency of membrane proteins in aqueous solution, it is difficult to simulate the membrane like environment to maintain the correct conformation of membrane proteins in vitro, which results in the slower-growing research on the structure and function of membrane proteins and related ligand drugs than that of water-soluble proteins. Membrane protein stabilization technology is the premise of establishing high specificity, high sensitivity and high throughput drug screening methods for membrane protein ligands, which is of great significance. In this paper, some techniques for stable separation and purification of membrane proteins are reviewed, including detergents, artificial membranes, polymers, lentiviral particles and so on, as well as their specific applications in drug screening.
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The paper aims to compare the protective effect of Salvia miltiorrhiza and Anemarrhena asphodeloides on AD cell model and investigate its protective mechanism by cell metabolomics platform. AD cell model was established by the abnormal phosphorylation of Tau protein in SH-SY5Y cells induced by okadaic acid. The protective effect of the extract of Salvia miltiorrhiza and Anemarrhena asphodeloides on the model was evaluated by cell proliferation-toxicity experiment. The metabolomics platform was used to study the efficacy of Salvia miltiorrhiza and Anemarrhena asphodeloides comprehensively, explore the potential biomarkers related to AD and the effect of drugs on the potential biomarkers. Salvia miltiorrhiza extract had a certain protective effect on the AD model (P < 0.05), while the Anemarrhena asphodeloides extract had no significant protective effect (P > 0.05). 45 significant differential metabolites and the related 12 metabolic pathways were identified using UHPLC-QTOF/MS platform, which were related to the AD cell model. After administration of Salvia miltiorrhiza extract, 30 different metabolites appeared callback, while after intervention of Anemarrhena asphodeloides extract, 7 metabolites appeared callback. The results showed that the extracts of Salvia miltiorrhiza and Anemarrhena asphodeloides had certain protective effects on the AD cell model with Tau protein abnormal phosphorylation, but Salvia miltiorrhiza had more extensive targets and could significantly improve the cell viability. The mechanism may be related to the regulation of the metabolic pathways of AD cell model induced by okadaic acid.
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ABC transporters on the intestinal barrier, blood-brain barrier and on tumor cells will affect drug bioavailability, transport across the blood-brain barrier and multidrug resistance. The active ingredients of traditional Chinese medicines can affect the function and expression of ABC transporters. When combined with pharmaceuticals the potential interaction between the two can change the efficacy of the medicines. We review the ABC transporter superfamily and their distribution with regard to their relationship and interactions with traditional Chinese medicine on the intestinal barrier and the blood-brain barrier, as well as their role in tumor multidrug resistance mediated by ABC transporters. We summarize the research progress over the past five years.
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Drug screening against Candida albicans has become more urgent due to the increasing incidence of infection and the development of drug-resistant strains. The microfluidic chip technique has shown great potential for high-throughput drug screening. In this study we developed a concentration gradient microfluidic chip platform for drug screening against Candida albicans. The generated concentration gradient on this platform was investigated qualitatively by monitoring the distribution of the fluorescent tracer fluorescein sodium and quantitatively by following the distribution of the model drug fluconazole as analyzed by HPLC; the effect of different flow conditions on the concentration gradient were determined. The ratio of the two aqueous phase flow rates was determined in the subsequent drug screening studies. Alamar Blue, an indicator of cell viability, was used in the susceptibility test for amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, terbinafine, 5-fluorocytosine and caspofungin as carried out on the established chip platform. The MIC range of the drugs, which was consistent with the MIC values of the CLSI-recommended standard, were obtained quickly and efficiently through the use of this platform, indicating that this new platform can quickly screen a series of antibacterial drugs in one run. In addition, the strain of Candida albicans we used showed resistance to terbinafine in our platform assay, consistent with the results of a 96-well plate assay, indicating that the platform can also be used for rapid screening of resistant strains.
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The blood-brain barrier (BBB) not only maintains the stability of the environment within the central nervous system by controlling the transport of substances on both sides of the blood and brain, but also plays an important role in the R&D of new drugs for neurological disorders. The establishment of an in vitro high-fidelity model to study BBB function is imperative for assessing barrier permeability of drugs and xenobiotics. However, the complexity of the BBB structure makes it difficult to replicate with an in vitro model. Compared to the traditional in vitro BBB model, the BBB-on-chip provides certain advantages in miniaturizing the system, reducing the amount of cells and medium required, and allowing simultaneously induction of shear stress. We review here the BBB-on-chip models from their establishment and characterization to applications in research of neuroinflammation, brain tumor and drug evaluation.
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A droplet microfluidic chip system was developed for drug screening against Candida albicans. The microfluidic chip was designed and prepared for the formation of droplets. Alamar blue was selected as an indicator for its characteristic of fluorescence mission in live cells. Four antifungal drugs (amphotericin B, caspofungin, 5-fluorocytosine, terbinafine) and a new drug (iodiconazole) were selected as model drugs to test the microfluidic chip approach. At the same time, 96-well microplate method was performed to verify the applicability of the chip method. The results showed that the developed droplet microfluidic chip platform was able to complete the antifungal susceptibility test within 2 h. In comparison with the 96-well microplate method, the microfluidic chip method showed a consistence of 100% with regard to the minimum inhibition concentrations and less reagent consumption. The new droplet microfluidic chip method is simple, rapid and suitable for rapid screening of antifungal drugs.
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Tanreqing injection (TRQ), a well-known traditional Chinese medicine formula, is commonly used to treat respiratory diseases. In the present study, a rapid, selective, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determinate the plasma contents of 5 major constituents of TRQ, including chlorogenic acid (CHA), caffeic acid (CFA), baicalin (BA), ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) in rats after intravenous administration of TRQ. Chromatographic separation was performed on an Agilent Zorbax SB-C column (3.5 μm, 100 mm × 2.1 mm), with acetonitrile and 0.1% aqueous formic acid as mobile phase at a flow rate of 0.3 mL·min. The calibration curves were linear over the ranges of 27.0-13 333.0 ng·mL for CFA, 30.0-14 933.0 ng·mL for CHA, 50.0-50 333.0 ng·mL for BA, 550.0-55 000.0 ng·mL for UDCA, and 480.0-48 000.0 ng·mL for CDCA, respectively. Intra- and inter-day precisions (relative standard deviations, RSDs) were from 3.11% to 14.08%. The extraction recoveries were greater than 71% and accuracy (relative recovery) was from 89% to 137% for all analytes, except endogenous bile acids. This validated method was successfully applied to the first pharmacokinetic study of CFA, CHA, BA, UDCA and CDCA in rat plasma after intravenous administration of TRQ.
Тема - темы
Animals , Male , Rats , Chromatography, High Pressure Liquid , Methods , Drugs, Chinese Herbal , Chemistry , Pharmacokinetics , Molecular Structure , Rats, Sprague-Dawley , Tandem Mass Spectrometry , MethodsРеферат
An offline two-dimensional system combining a rat cardiac mascle cell membrane chromatography time-of-flight mass spectrometry （CMC-TOF/MS） with a high performance liquid chromatography time-of-flight mass spectrometry （HPLC-TOF/MS） was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli. On the basis of the analysis of the first dimension, retention components of the urine sample were collected into 30 fractions （one fraction per minute）. Then offline analysis of the second dimension was carried out. 34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine, and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method. As a result, binding affinities decreased along with the process of deacylation, debenzoylation and demethylation, which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism. Moreover, some minor components in rat urine （Songorine, 14-benzoylneoline, Deoxyaconitine, etc. ） exerted relatively strong affinity on cell membranes are worth exploring. The results delivered by the system suggest that the CMC can be applied to in vivo study.
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Microbial metabolomics is a subject that chiefly studying all the low molecular weight metabolites in an organism or cells during their growing process. The progress of analytical technology promotes microbial metabolomics to make advancement. In this paper, the commonly used analytical technology, sample preparation and its application were discussed and the prospects of the analytical methods were also discussed.
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<p><b>AIM</b>To determine calycosin-7-O-beta-D-glucoside, astragaloside IV and formononetin in Radix Astragali and other relative samples by HPLC-MS.</p><p><b>METHODS</b>HPLC was carried out with Agilent 1100LC/MSD, equipped with Agilent Zorbax SB C18 column (250 mm x 4.6 mm ID, 5 microm) and mass spectrum detector. The mobile phase (CH3CN-H2O) was eluted in gradient mode.</p><p><b>RESULTS</b>The calibration curves of calycosin-7-O-beta-D-glucoside, astragaloside IV and formononetin were linear in the range of 0.03 - 1.21 microg x mL(-1), 0.35 - 13.86 microg x mL(-1) and 0.38 - 15.22 microg x mL(-1), respectively. These recoveries of samples were from 95% to 105% with RSD less than 1.5%.</p><p><b>CONCLUSION</b>The method was employed to analyse 25 samples of Radix Astragali and other relative samples, including Radix Astragali slice, Radix Astragali Preparata, Hedysarum polybotrys Hand. -Mazz, Astragalus ernestii Comb. The contents of three constituents vary greatly because of the species, place of collection and season of harvesting. This method could apply to evaluate the quality of Radix Astragali and it is simple, sensitive and reliable.</p>
Тема - темы
Astragalus Plant , Chemistry , Astragalus propinquus , Chemistry , China , Chromatography, High Pressure Liquid , Methods , Ecosystem , Glucosides , Isoflavones , Plant Roots , Chemistry , Plants, Medicinal , Chemistry , Reproducibility of Results , Saponins , Seasons , Species Specificity , Spectrometry, Mass, Electrospray Ionization , Methods , TriterpenesРеферат
<p><b>AIM</b>To investigate the stereoselective pharmacokinetic process of tetrahydropalmatine (THP) in rats.</p><p><b>METHODS</b>The concentrations of tetrahydropalmatine enantiomers in rat plasma were determined by coupled achiral and chiral HPLC method. The differences in plasma concentrations and pharmacokinetic parameters between the two enantiomers were compared by paired t-test.</p><p><b>RESULTS</b>The plasma levels of l-THP were always higher than those of d-THP in eight rats. There was significant difference between the main pharmacokinetic parameters of the two enantiomers.</p><p><b>CONCLUSION</b>Tetrahydropalmatine showed significant stereoselective pharmacokinetics in rats after an ig dose of the racemate.</p>
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Animals , Female , Male , Rats , Area Under Curve , Berberine Alkaloids , Chemistry , Pharmacokinetics , Chromatography, High Pressure Liquid , Methods , Corydalis , Chemistry , Molecular Structure , Plants, Medicinal , Chemistry , Rats, Sprague-Dawley , StereoisomerismРеферат
<p><b>OBJECTIVE</b>The chemical components of essential oil from fruit of Eucalyptus globulus were analyzed by GC-MS.</p><p><b>METHOD</b>The essential oil were extracted by steam distillation, then separated by capillary gas chromatography. The amount of the component from essential oil were determined by normalization methods. Chromatographic conditions were: capillary column DB-WAX (0.32 mm x 30 m, 0.25 microm) was used, column temperature: initial temperature at 40 degrees C for 3 min,ramping 5 C x min(-1) to 250 degrees C (holding for 10 min) the detector MS.</p><p><b>RESULT</b>31 component from the fruit of E. globulus were identified, which accounted for over 93.7% of total volatile oil.</p><p><b>CONCLUSION</b>The methods is reliable, stabilize and can be applied to identify the volatile oil from the fruit of E. globulus.</p>
Тема - темы
Cyclohexanols , Eucalyptus , Chemistry , Fruit , Chemistry , Gas Chromatography-Mass Spectrometry , Monoterpenes , Oils, Volatile , Chemistry , Plants, Medicinal , Chemistry , Sesquiterpenes , TerpenesРеферат
Objective: To establish capillary zone electrophoresis method for determination of sildenafil citrate (Viagra) content in its troche. Methods： Using tetrandrine as internal standard(IS), the electrophoretic separation was achieved with 25 mmol/L borate (pH=7.89) running buffer. And a voltage of 14 kV was applied to the 40 cm×75 μm(i.d) capillary. The analytes were introduced into capillary by siphon (1 s) and determined with on-column monitoring at 214 nm. Results：The determination could be completed within 4 min and the minimum concentration of detection was 5 μg/ml.The analytical results of sildenafil citrate samples demonstrated a good linear relationship within the range of 24-480 μg/ml.The relative standard deviations (RSD) of intra-day was less than 1.58% and that of inter-day was less than 2.46%.The present recoveries were between 95%-105％. Conclusion：The CZE method is accurate, simple, rapid and reliable, so it can be applied to the determination of sildenafil citrate content.
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Objective: To establish capillary zone electrophoresis method for determination of sildenafil citrate (Viagra) content in its troche. Methods： Using tetrandrine as internal standard(IS), the electrophoretic separation was achieved with 25 mmol/L borate (pH=7.89) running buffer. And a voltage of 14 kV was applied to the 40 cm×75 μm(i.d) capillary. The analytes were introduced into capillary by siphon (1 s) and determined with on-column monitoring at 214 nm. Results：The determination could be completed within 4 min and the minimum concentration of detection was 5 μg/ml.The analytical results of sildenafil citrate samples demonstrated a good linear relationship within the range of 24-480 μg/ml.The relative standard deviations (RSD) of intra-day was less than 1.58% and that of inter-day was less than 2.46%.The present recoveries were between 95%-105％. Conclusion：The CZE method is accurate, simple, rapid and reliable, so it can be applied to the determination of sildenafil citrate content.