LC-MS/MS analysis of puerarin and 18ß-glycyrrhetinic acid in human plasma after oral administration of Samso-eum and its application to pharmacokinetic study.

The aim of this study was to confirm pharmacokinetic screening of multiple components in healthy Korean subjects after oral administration of Samso-eum and perform quantitation of active components in the human plasma. Thirteen potential bioactive components [puerarin (PRR), daidzin, nodakenin, ginsenoside Rb1, 18ß-glycyrrhetinic acid (18ß-GTA), 6-shogaol, naringin, glycyrrhizin, hesperidin, platycodin D, naringenin, hesperetin, and 6-gingerol] were screened based on literature. The results showed that three analytes (daidzin, naringenin, and hesperetin) were detected in trace amounts. In addition, PRR and 18ß-GTA were detected in human plasma after the oral administration of Samso-eum. In this study, a liquid chromatography-electrospray ionization-tandem mass spectrometry method was validated for the simultaneous determination of PRR and 18ß-GTA in human plasma. This was the first study to evaluate pharmacokinetics of PRR and 18ß-GTA after the usual oral dose of Samso-eum (30 g containing 102.48 mg PRR, 48.18 mg glycyrrhizin) in human subjects.

Probing function and structure of trehalose-6-phosphate phosphatases from pathogenic organisms suggests distinct molecular groupings.

The trehalose biosynthetic pathway is of great interest for the development of novel therapeutics because trehalose is an essential disaccharide in many pathogens but is neither required nor synthesized in mammalian hosts. As such, trehalose-6-phosphate phosphatase (TPP), a key enzyme in trehalose biosynthesis, is likely an attractive target for novel chemotherapeutics. Based on a survey of genomes from a panel of parasitic nematodes and bacterial organisms and by way of a structure-based amino acid sequence alignment, we derive the topological structure of monoenzyme TPPs and classify them into 3 groups. Comparison of the functional roles of amino acid residues located in the active site for TPPs belonging to different groups reveal nuanced variations. Because current literature on this enzyme family shows a tendency to infer functional roles for individual amino acid residues, we investigated the roles of the strictly conserved aspartate tetrad in TPPs of the nematode Brugia malayi by using a conservative mutation approach. In contrast to aspartate-213, the residue inferred to carry out the nucleophilic attack on the substrate, we found that aspartate-215 and aspartate-428 of BmTPP are involved in the chemistry steps of enzymatic hydrolysis of the substrate. Therefore, we suggest that homology-based inference of functionally important amino acids by sequence comparison for monoenzyme TPPs should only be carried out for each of the 3 groups.-Cross, M., Lepage, R., Rajan, S., Biberacher, S., Young, N. D., Kim, B.-N., Coster, M. J., Gasser, R. B., Kim, J.-S., Hofmann, A. Probing function and structure of trehalose-6-phosphate phosphatases from pathogenic organisms suggests distinct molecular groupings.

Assessment of Choroidal Thickness Inside and Outside of Vascular Arcade in Diabetic Retinopathy Eyes Using Spectral-Domain Optical Coherence Tomography.

This study aimed to characterise the distribution of choroidal thickness (CT) in diabetic retinopathy eyes, inside and outside of the vascular arcade, as well as at the fovea, using spectral-domain optical coherence tomography (OCT). Forty-nine healthy eyes, 80 diabetic retinopathy (DR) eyes (59 non-proliferative diabetic retinopathy (NPDR) eyes and 21 proliferative diabetic retinopathy (PDR) eyes) were examined with OCT to obtain nine horizontal lines (far superotemporal, near superotemporal, central, near inferotemporal, far inferotemporal, far superonasal, near superonasal, near inferonasal, far inferonasal) inside and outside of the vascular arcade. Nine points were chosen in 0.5-mm intervals to calculate CT, which was measured at 81 points in each patient. In the DR group, CT decreased significantly, compared with the control group, in all nine horizontal lines except central and near inferotemporal (-29.74 to -36.97 µm, p < 0.05 for all). In the PDR group, CT decreased compared with the NPDR group, in all nine horizontal lines (-6.18 µm to -34.58 µm), but this difference was not significant. In DR eyes, an overall significant reduction of CT was observed inside and outside of the vascular arcade; CT showed a non-significant decrease in PDR eyes, compared with NPDR eyes.

Development of Simultaneous Analysis of Thirteen Bioactive Compounds in So-Cheong-Ryong-Tang Using UPLC-DAD.

So-Cheong-Ryong-Tang, which is a standardized Korean medicine of the National Health Insurance, is a traditional prescription for the treatment of allergic rhinitis, bronchitis, and bronchial asthma. Simultaneous analysis and development of SCRT is essential for its stability, efficacy, and risk management. In this study, a simple, reliable, and accurate method using ultrahigh-performance liquid chromatography (UPLC) fingerprinting with a diode array detector (DAD) was developed for the simultaneous analysis. The chromatographic separation of the analytes was performed by an ACQUITY UPLC BEH C18 column (1.7 µM, 2.1 × 100 mm, Waters) with a mobile phase of water containing 0.01% (v/v) phosphoric acid and acetonitrile containing 0.01% (v/v) phosphoric acid. The flow rate and detection wavelength were set at 0.4 mL/min and 215, 230, 254, and 280 nm. All calibration curves of the thirteen components showed good linearity (R2 > 0.999). The limit of detection and limit of quantification ranged 0.001-0.360 and 0.004-1.200 µg/mL, respectively. The relative standard deviation (RSD) of intra- and interday was less than 2.60%, and the recoveries were within the range 76.08-103.79% with an RSD value of 0.03-1.50%. The results showed that the developed method was simple, reliable, accurate, sensitive, and precise for the quantification of bioactive components of SCRT.

Crystal structure of an ASCH protein from Zymomonas mobilis and its ribonuclease activity specific for single-stranded RNA.

Activating signal cointegrator-1 homology (ASCH) domains were initially reported in human as a part of the ASC-1 transcriptional regulator, a component of a putative RNA-interacting protein complex; their presence has now been confirmed in a wide range of organisms. Here, we have determined the trigonal and monoclinic crystal structures of an ASCH domain-containing protein from Zymomonas mobilis (ZmASCH), and analyzed the structural determinants of its nucleic acid processing activity. The protein has a central ß-barrel structure with several nearby α-helices. Positively charged surface patches form a cleft that runs through the pocket formed between the ß-barrel and the surrounding α-helices. We further demonstrate by means of in vitro assays that ZmASCH binds nucleic acids, and degrades single-stranded RNAs in a magnesium ion-dependent manner with a cleavage preference for the phosphodiester bond between the pyrimidine and adenine nucleotides. ZmASCH also removes a nucleotide at the 5'-end. Mutagenesis studies, guided by molecular dynamics simulations, confirmed that three residues (Tyr47, Lys53, and Ser128) situated in the cleft contribute to nucleic acid-binding and RNA cleavage activities. These structural and biochemical studies imply that prokaryotic ASCH may function to control the cellular RNA amount.