| Cerebralcare Granule(CG)is developed from the TCM "Si Wu Tang" and is composed of eleven herbs.CG,which has multiple pharmacological activities,including nourishing yin and tonifying blood,suppressing hyperactive liver and subsiding yang,promoting blood circulation to remove meridian obstruction,has been mainly used for the clinical treatment of cerebrovascular diseases including stroke,headache and dizziness.It is remarkable to the clinical curative effective of CG,but still there is much space for further study of pharmacodynamic substance foundation,action mechanism and quality control.On the basis of overall concept is deemed as the guiding ideology in this study,multiple analytical technologies are employed for the investigation of the material foundation and the quality control methods of CG.1.The methods of quality evaluation and chemical components identification of CG280 nm UPLC digitalized fingerprints of 30 batches of CG samples were developed and systematic fingerprints quantification method was employed to assess the results to reveal the feature of CG fingerprints.UPLC/Q-TOF-MS coupled with UPLC/IT-MSn method were developed for carrying out the comprehensive characterization of the 64 common peaks of CG.Besides,the ascription of each components identified in the decoction to their derived medicinal materials was investigated.Meanwhile,fingerprints coupled with chemometric analysis provide a novel strategy for the rapid identification of analytical markers for assessing the quality of CG.A simultaneously quantitative method of 8 active components was developed via UPLC,which is rapid and reliable and can be an appropriate means of quality control of TCMs with botanical drugs.2.Analysis of the constituents in rat plasma and the pharmacokinetic study of the main component after oral administration of CGThe effective constituents in rats plasma after oral administrated of CG was analyzed by UPLC/Q-TOF-MS,compared with the blank plasma,medicine in vitro and the extract of CG,and the ownership of every peak was analyzed.28 original constituents came from 8 medicine were detected in the rats plasma,and including organic acids(7),glucosides(3),alkaloids(9)and anthraquinones(8).An ultra fast liquid chromatography-tandem mass sepectrometry(UFLC-MS/MS)method was developed for simultaneous determination of seven active alkaloid components in rat plasma after oral administration of Cerebralcare Granule.Plasma samples were pretreated by protein precipitation with acetronitrile containing the internal standard diazepam.The detection was performed on an electrospray ionization triple quadrupole tandem mass spectrometer using multiple reaction monitoring(MRM)with positive ionization mode.Chromatographic separation was achieved on a Phenomenex Kinetex C18 column(100×2.1 mm,2.6 m)with gradient elution using mobile phase consisting of acetonitrile-0.1%formic acid in water at a flow rate of 0.3 mL/min.The established method was fully validated and proved to be sensitive and specific with lower limits of quantification(LLOQs)all less than 0.02 ng/mL in rat plasma.Good linearities of seven alkaloids were obtained in respective concentration ranges(r>0.9923).The intra-and inter-day precisions were below of 15%for all the seven alkaloids in terms of relative standard deviation(RSD),and the accuracies were ranged from-2.7%to 8.3%in terms of relative error(RE).Extraction recovery,matrix effect and stability were within the required limits in rat plasma.Following oral administration of CG?double peaks were observed in both individual and mean plasma concentration profiles of seven alkaloids.The results might be presumably due to the different physiochemical properties of each compound or the pharmacokinetic interaction of the prescribed chemical components.A rapid analytical method for determining 3 organic acids,4 glucosides and 3 anthraquinones in rat plasma by UPLC-MS/MS was established.Plasma samples were pretreated by protein precipitation with acetronitrile containing the internal standard chloramphenicol.Detection was performed by MRM mode using ESI in negative mode for those 10 components.The established method was fully validated and proved to be sensitive and specific with lower limits of quantification(LLOQs)all less than 0.03 ng/mL in rat plasma.Good linearities of these 10 compounds were obtained in respective concentration ranges(r>0.9923).The intra-day and inter-day precisions(RSD)at three QC levels were both less than 15%and the accuracies ranged from-9.2%to 14%.Extraction recovery,matrix effects were within the required limits in rat plasma.All the 10 analytes were fitted with a two-compartment model after oral administration of CG,and slowly absorbed rapidly within 0.88 h.The validated method was successfully applied to investigate the pharmacokinetics of 10 compounds in rat plasma after oral administration of CG.These works could provide more in-depth insights into these active compounds working in vivo and would be helpful for further revealing the pharmacology and mechanisms of CG.3.Study of the CG nerve cell protection effect on zebrafish modelTo investigate the biopotency of CG nerve cell protection effect by establishing a zebrafish central nerve injury model.A high-throughput screen for zebrafish central nerve injury proteins can be performed by a combined use of iTRAQ and Nano-LC-MS/MS and to preliminarily explore the molecular mechanism after the treatment of CG.The results indicate that CG has excellent nerve cell protection effect.The significant and dose-dependent apoptosis of nerve cells observed in the CG concentrated with 1000 ?g/mL that effective responsible for its action of nerve protection,and the efficacy was(59.0±3.54)%.With the high throughput proteomic technology of isobaric tags for relative and absolute quantitation(iTRAQ)-coupled with Nano-LC-MS/MS,1,933 unique proteins were identified,and 130 proteins showed significant changes.These proteins have roles in biological processes most represented by proteins detected in the zebrafish neuroendocrine brain includes translation,metabolic process and Neuronal ion channel clustering.Efl-?(elongation factor 1-alpha)and?-6-F(Na+/K+transporting ATPase alpha 1 polypeptide)were validated by Western blotting.Two sets of data between a statistically significant difference(p<0.05).These results were consistent with those from quantitative mass spectrometry.4.Zebrafish-Based Biological Quality Control for Neuroprotective Efficacy of Cerebralcare GranuleBiological activity effect and stability of CG were quantitatively assessed in a zebrafish central nerve injury model which was established by using the CNS neurotoxic revulsant mycophenolate mofetil(MMF).Central nerve injury zebrafish model as a biological quality control standard for the CG nerve cell protection exhibited excellent precision(intra-day RSD 2.2-3.7%,inter-day RSD 1.2%),reproducibility(RSD 4.9%)and stability(RSD 8.4%);In the range of 100.0-1000 ?g/mL there was a significant linear correlation(P%=0.082C+4.439,r=0.9950);and the method met the requirements of the biological activity determination of guiding principles.Zebrafish central nerve injury model-based biological quality control for CG nerve cell protection methods is a rapid,reliable and reproducible method,and can be used as a useful supplement in quality control,and it could provide some technical information for quality control of other traditional Chinese medicine. |
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