Yonggui Song

Validated rapid resolution LC-ESI–MS/MS method for simultaneous determination of five pulchinenosides from Pulsatilla chinensis (Bunge) Regel in rat plasma: Application to pharmacokinetics and bioavailability studies

A simple, RRLC-ESI-MS/MS method was developed for the simultaneous determination of five oleanane pulchinenosides (B3, BD, B7, B10, and B11), in rat plasma following solid-phase extraction (SPE). Detection and quantitation were performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) mode. The MS/MS transitions of the triterpenoidal saponins: m/z 911.4→603.2, 749.4→471.3, 895.6→733.2, 733.5→455.3, and 579.3→371.1 were monitored for B3, BD, B7 and B10, B11 and internal standard (Forsythin), respectively. The method was validated in plasma samples, showed good linearity over a wide concentration range (r(2)>0.99), and with lower limits of quantification of 1.11 (B3), 0.751 (BD), 0.996 (B7), 0.415 (B10), and 0.332 (B11)ng/mL. The intra- and inter-day assay variability was less than 15% for all analytes. The mean extraction recoveries of analytes and IS from rats plasma were all more than 70.0%. The validation results demonstrate that this method is robust and specific. The validated method was successfully applied for the pharmacokinetic and bioavailability studies of the five pulchinenosides which are potentially active saponins present in P. chinensis saponins (PRS) extracts.

Differential pharmacokinetics and the brain distribution of morphine and ephedrine constitutional isomers in rats after oral administration with Keke capsule using rapid‐resolution LC–MS/MS

Opioid and ephedra alkaloids known as the active ingredients for Keke capsule, which is used to treat coughs and bronchial asthma, could have potential adverse effects on the central nervous system. Therefore, an efficient, sensitive rapid-resolution LC-MS/MS method for the simultaneous determination of morphine, ephedrine, and pseudoephedrine in rat plasma and brain tissue homogenate has been developed. The method was validated in the plasma and brain tissue samples, showed good linearity over a wide concentration range (r(2) > 0.99). The intra- and interday assay variability was less than 15% for all analytes, and the accuracy was between -8.8 and 5.7%. The study provided the pharmacokinetics profiles and the brain regional distribution of the three active alkaloids after oral administration of Keke capsule. The results also indicated that significant difference in pharmacokinetics parameters of the epimers was observed between ephedrine and pseudoephedrine.

Antiviral herbs--present and future.

Viral disease is a calamity which absolutely can not be ignored for human health. The emergence of drug resistance and spread of new virus will be the new challenge against viral disease. To find and develop new antivirus agents with properties of safety, significant effect and low toxicity is the pressing question facing humans today. Because of its advantages, including rich resources, low price, less adverse effect, Traditional Chinese medicine (TCM) have become the research focus in antiviral treatment. In recent years, there are numerous articles about the studies from separation of active ingredients to the antiviral mechanism. In this paper, the progress in experimental study was illustrated on the basis of active ingredients, species of virus, mechanism, clinical application. Obviously, TCM have obvious advantages in the treatment of virus infectious disease and has a broad prospect of application.

New metabolites of acteoside identified by ultra-performance liquid chromatography/quadrupole-time-of-flight MSE in rat plasma, urine, and feces

Acteoside, which belongs to the family of phenylethanoid glycosides (PhGs), has extensive biological activities, including strong antioxidant, anti-inflammatory, hepatoprotect, and cell apoptosis regulation. Like other PhGs compounds, the fate of acteoside in the gut for both parent polyphenols and their degradation products, small phenolic acid and aromatic catabolites cannot be ignored. Therefore, in this work, expanded and systematical investigation for metabolism characteristic profiles of acteoside in vivo by ultra-performance liquid chromatography/ quadrupole-time-of-flight and a new MS(E) data collection technology had been studied. This was equivalent to non-slective MS/MS scans and helpful to explore new metabolites. After oral administration of 200mg/kg acteoside, He et al. (2011) a total of 44 metabolites was detected and identified, and 37 of them were reported for the first time. Among them, 35 were parent drug metabolites classified in 14 groups. Owen et al. (2003) Through the comprehensive metabolites study in plasma, urine and feces, acteoside systemical metabolites profiles and characteristics elaborated firstly. The relative content of metabolites research showed that acteoside could exist stably and the process for biotransformation of acteoside in blood keep extreme short time. Pan and Hori (1996) The significant new transformation of isomerization from acteoside to isoacteoside had been firstly found and confirmed. The results of this work provided new information for the clarification of the metabolism of acteoside and rendered a very valuable theoretical basis for the development of novel ideal dosage forms of acteoside in the future.

Early Growth Response Protein-1 Involves in Transforming Growth factor-β1 Induced Epithelial-Mesenchymal Transition and Inhibits Migration of Non-Small-Cell Lung Cancer Cells.

The zinc finger transcription factor EGR1 has a role in controlling synaptic plasticity, wound repair, female reproductive capacity, inflammation, growth control, apoptosis and tumor progression. Recent studies mainly focused on its role in growth control and apoptosis, however, little is known about its role in epithelial-mesenchymal transition (EMT). Here, we aim to explore whether EGR 1 is involved in TGF-β1-induced EMT in non-small- cell lung cancer cells. Transforming growth factor (TGF)-β1 was utilized to induce EMT in this study. Western blotting, RT-PCR, and transwell chambers were used to identify phenotype changes. Western blotting was also used to observe changes of the expression of EGR 1. The lentivirus-mediated EGR 1 vector was used to increase EGR1 expression. We investigated the change of migration to evaluate the effect of EGR 1 on non-small-cell lung cancer cells migration by transwell chambers. After stimulating with TGF-β1, almost all A549 cells and Luca 1 cells (Non-small-cell lung cancer primary cells) changed to mesenchymal phenotype and acquired more migration capabilities. These cells also had lower EGR 1 protein expression. Overexpression of EGR 1 gene with EGR 1 vector could decrease tumor cell migration capabilities significantly after adding TGF-β1. These data showed an important role of EGR 1 in the EMT of non-small-cell lung cancer cells, as well as migration.

An integrated strategy using UPLC–QTOF-MS E and UPLC–QTOF-MRM (enhanced target) for pharmacokinetics study of wine processed Schisandra Chinensis fructus in rats

HIGHLIGHTSDevelopment of an integrated strategy for extensive pharmacokinetics assessments.Multi‐components analysis based on the use of UPLC‐TOF‐MSE and UPLC‐TOF‐MRM.Application for the simultaneous determination of 12 lignans in rat plasma with or without authentic standards.Significant differences in pharmacokinetics parameters of lignans was observed between schizandrin and gomisin compounds. ABSTRACT Currently the pharmacokinetic (PK) research of herbal medicines is still limited and facing critical technical challenges on quantitative analysis of multi‐components from biological matrices which often accompanied by lacking of authentic standards and low concentration. This present work contributes to the development of an integrated strategy for extensive pharmacokinetics assessments, and a selective and sensitive method independent of authentic standards for multi‐components analysis based on the use of ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight/MSE (UPLC‐TOF‐MSE) and UPLC‐TOF‐MRM (rnhanced target). Initially, phytochemicals were identified by UPLC‐TOF‐MSE analysis, subsequently the identified components were matched with authentic standards and pre‐classified, and UPLC‐QTOF‐MRM method optimized and developed. To guarantee reliable results, three rules are necessary: (1) detection with a mass error of less than 5 ppm; (2) same class chemical compositions with structural high similarity between analytes with and without authentic reference substance; (3) a matching retention time between TOF‐MRM mode and TOF‐MSE within 0.2 min. The developed and validated method was applied for the simultaneous determination of 12 lignans in rat plasma after administered with wine processed Schisandra Chinensis fructus (WPSCF) extract. Such an approach was found capable of providing extensive pharmacokinetic profiles of multi‐components absorbed into blood after oral administrated with WPSCF extract. The results also indicated that significant difference in pharmacokinetics parameters of dibenzocyclooctadiene lignans was observed between schizandrin and gomisin compounds. For lignans, the absorption via gastrointestinal tract were all rapid and maintained relatively long retention time, especially for schisantherin A and schisantherin B with higher plasma exposure.

The different metabolism of morusin in various species and its potent inhibition against UDP-glucuronosyltransferase (UGT) and cytochrome p450 (CYP450) enzymes.

Abstract 1. The aim of this study was to investigate the inhibitory effect of morusin on Glucuronosyltransferase (UGT) isoforms and cytochrome P450 enzymes (CYP450s). We also investigated the metabolism of morusin in human, rat, dog, monkey, and minipig liver microsomes. 2. 100 μM of morusin exhibited strong inhibition on all UGTs and CYP450s. The half inhibition concentration (IC50) values for CYP3A4, CYP1A2, CYP2C9, CYP2E1, UGT1A6, UGT1A7, and UGT1A8 were 2.13, 1.27, 3.18, 9.28, 4.23, 0.98, and 3.00 μM, and the inhibition kinetic parameters (Ki) were 1.34, 1.16, 2.98, 6.23, 4.09, 0.62, and 2.11 μM, respectively. 3. Metabolism of morusin exhibited significant species differences. The quantities of M1 from minipig, monkey, dog, and rat were 7.8, 11.9, 2.0, and 6.3-fold of human levels. The Km values in HLMs, RLMs, MLMs, DLMs, and PLMs were 7.84, 22.77, 14.32, 9.13, and 22.83 μM, and Vmax for these species were 0.09, 1.23, 1.43, 0.15, and 0.75 nmol/min/mg, respectively. CLint (intrinsic clearance) values (Vmax/Km) for morusin obeyed the following order: monkey > rat > minipig > dog > human. CLH (hepatic clearance) values for humans, dogs, and rats were calculated to be 8.28, 17.38, and 35.12 mL/min/kg body weight, respectively. 4. This study provided vital information to understand the inhibitory potential and metabolic behavior of morusin among various species.

Endogenous L-Carnosine Level in Diabetes Rat Cardiac Muscle

A novel method for quantitation of cardiac muscle carnosine levels using HPLC-UV is described. In this simple and reliable method, carnosine from the rat cardiac muscle and the internal standard, thymopentin, were extracted by protein precipitation with acetonitrile. The method was linear up to 60.96 μg·mL−1 for L-carnosine. The calibration curve was linear in concentration ranges from 0.5 to 60.96 μg·mL−1. The relative standard deviations obtained for intra- and interday precision were lower than 12% and the recoveries were higher than 90% for both carnosine and internal standard. We successfully applied this method to the analysis of endogenous carnosine in cardiac muscle of the diabetes rats and healthy control rats. The concentration of carnosine was significantly lower in the diabetes rats group, compared to that in the healthy control rats. These results support the usefulness of this method as a means of quantitating carnosine and illustrate the important role of L-carnosine in cardiac muscle.

Comparative pharmacokinetic profiles of five poorly soluble pulchinenosides in different formulations from Pulsatilla chinensis saponins extracts for enhanced bioavailability.

Pulsatilla chinensis, a traditional Chinese medicine (TCM), has been used for treating amoebic diseases, vaginal trichomoniasis and bacterial infections over a long history. Now growing attention has been attracted to its antitumor activities. The purpose of this work was to compare the pharmacokinetic profiles of pulchinenosides in different formulations and to improve their oral bioavailability. Extracts of P. chinensis saponins were prepared for PRS-Na (salt forming), PRS-HPβCD (hydroxypropyl-β-cyclodextrin inclusion complex), PRS-O/W (oil-in-water emulsion) and PRS-silica (micronization), respectively. A simpler and more durable LC-MS/MS method was developed in this study for quantitative analysis of pulsatilla sapoin D, B7, B10, B11 and sapoin PD simultaneously. The four formulations enhanced saponins oral bioavailability to varying degrees, as PRS-HPβCD > PRS-silica > PRS-O/W > PRS-Na, which indicated that water-soluble preparations can obviously improve the solubility of saponins, and are helpful to increase bioavailability. In particular, hydroxypropyl-β-cyclodextrin inclusion complex was the most effective way to promote absorption of saponins, raising the F values (bioavailability) >20 times. Therefore, P. chinensis saponin molecules can be slowly released by emulsion and micronization, which can avoid the enormous Cmax appearing in HPβCD, considering the pharmacokinetics profiles. However, appropriate pharmacokinetic parameters were observed in PRS-Na, although the F value was minimum among the four preparations.

Identification of the Metabolic Enzyme Involved Morusin Metabolism and Characterization of Its Metabolites by Ultraperformance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (UPLC/Q-TOF-MS/MS)

Morusin, the important active component of a traditional Chinese medicine, Morus alba L., has been shown to exhibit many vital pharmacological activities. In this study, six recombinant CYP450 supersomes and liver microsomes were used to perform metabolic studies. Chemical inhibition studies and screening assays with recombinant human cytochrome P450s were also used to characterize the CYP450 isoforms involved in morusin metabolism. The morusin metabolites identified varied greatly among different species. Eight metabolites of morusin were detected in the liver microsomes from pigs (PLMs), rats (RLMs), and monkeys (MLMs) by LC-MS/MS and six metabolites were detected in the liver microsomes from humans (HLMs), rabbits (RAMs), and dogs (DLMs). Four metabolites (M1, M2, M5, and M7) were found in all species and hydroxylation was the major metabolic transformation. CYP1A2, CYP2C9, CYP2D6, CYP2E1, CYP3A4, and CYP2C19 contributed differently to the metabolism of morusin. Compared to other CYP450 isoforms, CYP3A4 played the most significant role in the metabolism of morusin in human liver microsomes. These results are significant to better understand the metabolic behaviors of morusin among various species.