Dazheng Zhou

Salvianolic Acids for Injection (SAFI) Suppresses Inflammatory Responses in Activated Microglia to Attenuate Brain Damage in Focal Cerebral Ischemia.

BACKGROUND Inflammatory reactions induced by microglia in the brain play crucial roles in ischemia/reperfusion (I/R) cerebral injuries. Microglia activation has been shown to be closely related to TLR4/NF-κB signal pathways. Salvianolic acids for injection (SAFI) have been used in clinical practice to treat ischemic stroke with reported neuroprotective effects; however, the underlying mechanisms are still uncertain. OBJECTIVE AND METHODS First, we studied the effect of SAFI on inflammatory responses in LPS-stimulated BV-2 microglia. Then, to discover whether the beneficial in vitro effects of SAFI lead to in vivo therapeutic effects, an MCAO (Middle cerebral artery occlusion) rat model was further employed to elucidate the probable mechanism of SAFI in treating ischemic stroke. Rats in the SAFI group were given SAFI (23 or 46mg/kg) before I/R injury. RESULTS The results showed that SAFI treatment significantly decreased neuroinflammation and the infarction volume compared with the vehicle group. Activation of microglia cells was reduced, and TLR4/NF-κB signals, which were markedly inhibited by SAFI treatment in ischemic hemisphere, were accompanied by reduced expression and release of cytokines IL-1β and IL-6. CONCLUSION This study provides evidence that SAFI effectively protects the brain after cerebral ischemia, which may be caused by attenuating inflammation in microglia.

Salvianolic Acid Y: A New Protector of PC12 Cells against Hydrogen Peroxide-Induced Injury from Salvia officinalis

Salvianolic acid Y (TSL 1), a new phenolic acid with the same planar structure as salvianolic acid B, was isolated from Salvia officinalis. The structural elucidation and stereochemistry determination were achieved by spectroscopic and chemical methods, including 1D, 2D-NMR (1H-1H COSY, HMQC and HMBC) and circular dichroism (CD) experiments. The biosynthesis pathway of salvianolic acid B and salvianolic acid Y (TSL 1) was proposed based on structural analysis. The protection of PC12 cells from injury induced by H2O2 was assessed in vitro using a cell viability assay. Salvianolic acid Y (TSL 1) protected cells from injury by 54.2%, which was significantly higher than salvianolic acid B (35.2%).

Simultaneous Qualitative and Quantitative Analysis of Multiple Chemical Constituents in YiQiFuMai Injection by Ultra-Fast Liquid Chromatography Coupled with Ion Trap Time-of-Flight Mass Spectrometry

YiQiFuMai injection (YQFM) is a modern lyophilized powder preparation derived from the traditional Chinese medicine Sheng-mai san (SMS) used for treating cardiovascular diseases, such as chronic heart failure. However, its chemical composition has not been fully elucidated, particularly for the preparation derived from Ophiopogon japonicus. This study aimed to establish a systematic and reliable method to quickly and simultaneously analyze the chemical constituents in YQFM by ultra-fast liquid chromatography coupled with ion trap time-of-flight mass spectrometry (UFLC-IT-TOF/MS). Sixty-five compounds in YQFM were tentatively identified by comparison with reference substances or literature data. Furthermore, twenty-one compounds, including three ophiopogonins, fifteen ginsenosides and three lignans were quantified by UFLC-IT-TOF/MS. Notably, this is the first determination of steroidal saponins from O. japonicus in YQFM. The relative standard deviations (RSDs) of intra- and inter-day precision, reproducibility and stability were <4.9% and all analytes showed good linearity (R2 ≥ 0.9952) and acceptable recovery of 91.8%–104.2% (RSD ≤ 5.4%), indicating that the methods were reliable. These methods were successfully applied to quantitative analysis of ten batches of YQFM. The developed approach can provide useful and comprehensive information for quality control, further mechanistic studies in vivo and clinical application of YQFM.

Nontargeted metabolomics approach for the differentiation of cultivation ages of mountain cultivated ginseng leaves using UHPLC/QTOF-MS

&NA; The adulteration or falsification of the cultivation age of mountain cultivated ginseng (MCG) has been a serious problem in the commercial MCG market. To develop an efficient discrimination tool for the cultivation age and to explore potential age‐dependent markers, an optimized ultra high‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UHPLC/QTOF‐MS)‐based metabolomics approach was applied in the global metabolite profiling of 156 MCG leaf (MGL) samples aged from 6 to 18 years. Multivariate statistical methods such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS‐DA) were used to compare the derived patterns between MGL samples of different cultivation ages. The present study demonstrated that 6–18‐year‐old MGL samples can be successfully discriminated using two simple successive steps, together with four PLS‐DA discrimination models. Furthermore, 39 robust age‐dependent markers enabling differentiation among the 6–18‐year‐old MGL samples were discovered. The results were validated by a permutation test and an external test set to verify the predictability and reliability of the established discrimination models. More importantly, without destroying the MCG roots, the proposed approach could also be applied to discriminate MCG root ages indirectly, using a minimum amount of homophyletic MGL samples combined with the established four PLS‐DA models and identified markers. Additionally, to the best of our knowledge, this is the first study in which 6−18‐year‐old MCG root ages have been nondestructively differentiated by analyzing homophyletic MGL samples using UHPLC/QTOF‐MS analysis and two simple successive steps together with four PLS‐DA models. The method developed in this study can be used as a standard protocol for discriminating and predicting MGL ages directly and homophyletic MCG root ages indirectly. HighlightsA sensitive and reliable UHPLC/QTOF‐MS method was developed for the global metabolite profiling of Mountain Cultivated Ginseng (MCG) leaves.Application of UHPLC/QTOF‐MS‐based metabolomics approach together with multivariate statistical analysis for discriminating the cultivation age of MCG leaves ranging from 6 to 18 years and revealing 39 key age‐dependent markers.This is the first study in which 6–18‐year‐old MCG root ages have been nondestructively differentiated by analyzing homophyletic MCG leaves using UHPLC/QTOF‐MS analysis and two simple successive steps together with four PLS‐DA models.

Integrated pharmacokinetics of ginsenosides after intravenous administration of YiQiFuMai powder injection in rats with chronic heart failure by UFLC–MS/MS

YiQiFuMai powder injection (YQFM), derived from the classical traditional Chinese medicine (TCM) formula Shengmai San, is a modern preparation widely used to combat cardiovascular diseases, chronic heart failure (CHF) for example, in clinical practice in China. Ginsenosides are the major components of YQFM, which are responsible for its therapeutic effect. In this research, we developed a rapid, sensitive and simple method for simultaneous determination of ten ginsenosides from YQFM in CHF rat plasma with ultra-fast liquid chromatography tandem mass spectrometry (UFLC-MS/MS). After solid phase extraction (SPE), chromatography was done on an Acquity UPLC HSS T3 column (1.8μm, 100mm×2.1mm, i.d.) through an 8.0min gradient elution with acetonitrile and 0.1% formic acid in water, while mass spectrometry was performed in the positive ion electrospray ionization (ESI) mode. A good linearity was achieved for each analyte with correlation coefficient (r) >0.9920. The lower limits of quantification (LLOQ) were 1.25ng/mL for ginsenoside Rg1, Rd, Re and Rh1, 2.5ng/mL for ginsenoside Rf, Rg3, Rb2 and Rb3 and 5.0ng/mL for ginsenoside Rb1 and Rc, respectively. All the precision (RSD) data ranged from 1.7-14.5% and the accuracy (RE) data was within ±13.73%. Moreover, the validated method has been applied to investigate the integrated pharmacokinetic profiles of ginsenosides in CHF rats following intravenous administration of YQFM successfully.

A quality marker study on salvianolic acids for injection.

BACKGROUND The quality of Chinese medicine (CM) has being an active and challenging research area for CM. Prof. Chang-Xiao Liu et al first proposed the concept of quality marker (Q-Marker) for the quality evaluation and control on CM. This article describe the exploratory studies of Q-Marker in salvianolic acids for injection (SAI) based on this new concept. PURPOSE This study was designed to screen Q-Marker of SAI and establish its quality control method based on the concept of CM Q-Marker. METHODS Based on the concept of CM Q-Marker, the SAI was investigated for the identification of chemical components and their sources. The pharmacological effects on cerebral ischemia and reperfusion induced injury in rats were also investigated. Furthermore, the target cell extracts and pharmacokinetic studies were conducted to screen Q-Markers. Finally, the fingerprints and determination based on Q-Markers were established to assess the quality of SAI more effectively. RESULTS Overall, 20 constituents in SAI were identified. It was found that salvianolic acid B (SA-B), rosmarinic acid (RA), lithospermic acid (LA), salvianolic acid D (SA-D) and salvianolic acid Y (SA-Y) are major chemical components of SAI. Based on chemical components identifications, analysis of their sources, target cell extracts and pharmacokinetic studies, four phenolic acids, namely SA-B, RA, LA and SA-D, were screened and determined as effective Q-Markers of SAI. CONCLUSION This study demonstrated that the described method is a powerful approach for detecting Q-Markers, which can be used as control index for the quality assessment of CM.