| Background:Zige Freeze-dried Powder is a composition drug based on the traditional Chinese medicine (TCM) theory and the previous reports on the pharmacologic actions of catalpol and puerarin. In vitro, it have been certificated that the Zige Freeze-dried Powder can protect PC12cell from the damage induced by CoCl2and inhibit the apoptosis of the injured PC12cells, and displays remarkable activity in unit of "Brain Neurovascular unit" which is conditional on OGD. In vivo, the Zige Freeze-dried Powder also decreased the brain edema and improved behavioural disorders appeared12h after cerebral infaction.Although there are some reports on in vitro and in vivo investigation of Zige Freeze-dried Powder bioactivities, few studies focused on the pharmacokinetics and the biodistribution of Zige Freeze-dried Powder. The ability of Zige Freeze-dried Powder to penetrate blood brain barrier (BBB) is a pre-requisite for treating neurodegenerative diseases. So it is necessary to study the distribution and pharmacokinetics of catalpol Zige Freeze-dried Powder in CSF.Objectives:In the study, we evaluated the security of Zige Freeze-dried Powder based on the distribution of puerarin in the rat liver and kidney firstly, and then the pharmacokinetics and distribution of Zige Freeze-dried Powder were studied after intravenous administration (i.v.). Furthermore, we investigated the blood-brain barrier permeability of Zige Freeze-dried Powder, and then the mechanism of improving the blood-brain barrier permeability was studied. All the researches are beneficial to the further study of bioactive mechanisms and the development of therapy schedules of Zige Freeze-dried Powder.Methods and Results:1. Investigate the pharmacokinetics and biodistribution of catalpol and puerarin in the rat's plasma and CSFA high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method was developed and validated to investigate the pharmacokinetics and biodistribution of catalpol in the rats plasma and cerebrospinal fluid (CSF) and a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method was developed and validated to investigate the pharmacokinetics and biodistribution of puerarin in rats the plasma and CSF after i.v. Catalpol Freeze-dried Powder, Zige Freeze-dried Powder and Puerarin Freeze-dried Powder. The plasma and CSF samples were pretreated with methanol for protein precipitation and the supernatants were separated on a Diamonsil C18column (150mm×4.6mm,5μm) with the mobile phase consisting of10mM ammonium acetate in methanol and10mM ammonium acetate in water (50:50, v/v) for the analysis of catalpol and0.1%formic acid in methanol and10mM ammonium acetate in water (80:20, v/v) for the analysis of puerarin at a flow rate of0.6mL·min-1. Detection was performed on a triple-quadrupole tandem mass spectrometer under a multiple reaction-monitoring (MRM) mode using aucubin as the IS for the the analysis of catalpol and genistein for the analysis of puerarin. The calibration curves had good linear with r>0.990. The lower limit of quantification (LLOQ) were10ng·mL-1,20ng·mL-1for the analysis of catalpol in the plasma and CSF respectively and20ng·mL-1for the analysis of puerarin in the plasma and CSF.The intra-and inter-day precision were found to be less than15%, and the accuracy were ranged in±15%for the biosamples. The extraction recoveries of the catalpol and puerarin from the biosamples were similar to that of the IS. The developed and validated method was successfully applied to research the pharmacokinetics of catalpol and puerarin in rats plasma and CSF after i.v. Catalpol Freeze-dried Powder, Zige Freeze-dried Powder and Puerarin Freeze-dried Powder. The pharmacokinetic analyses of catalpol and puerarin in rat plasma and CSF were performed via the proprietary DAS2.1computer software package. The pharmacokinetic parameters may be beneficial to the further study of the bioactive mechanism and help in the clinical pharmacokinetic studies after i.v. administration of Zige Freeze-dried Powder.The results revealed that the contents of catalpol arrived the max values with23024.4ng·mL-1and415.5ng·mL-1, the t1/2were0.369h and1.022h and the values of AUC(0～5h) were10732.5ng·h·mL-1and395.4ng·h·mL-1, the values of MRT(0～∞h) were0.437h and1.357h in plasma and CSF respectively. The contents of puerarin arrived the max values with56529.4ng·mL-1and899.6ng·mL-1, the t1/2were0.311h and0.537h, the values of AUC(0～5h) were17136.3ng·h·mL-1and449.6ng·h·mL-1, the values of MRT(0～∞h) were0.273h and0.640h in plasma and CSF respectively The Tmax for catalpol and puerarin in CSF were0.050h and0.080h respectively. The AUCCSF/AUCplasma for catalpol and puerarin were3.7%and2.6%respectively. The results indicated catalpol and puerarin can be transported into BBB rapidly. The results will supply beneficial information to the clinical pharmacokinetic studies of Zige Freeze-dried Powder.2. Investigate the pharmacokinetics and biodistribution of catalpol and puerarin in the cerebral ischemia rats plasma and CSFThe developed and validated methods were successly used to determine the catalpol and puerarin in the plasma and CSF after i.v. of Zige Freeze-dried Powder in the cerebral ischemia rats. The results revealed that the contents of catalpol arrived the max values with17523.2ng·mL-1and592.2ng·mL-1, the t1/2were0.455h and0.860h and the values of AUC(0～5h) were7165.1ng·h·mL-1and516.3ng·h·mL-1, the values of MRT(0～∞h) were0.451h and1.110h in plasma and CSF respectively. The contents of puerarin arrived the max values with63243.0ng·mL-1and1812.2ng·mL-1, the t1/2were0.755h and0.593h, the values of AUC(0～5h) were20059.8ng·h·mL-1and1011.0 ng·h·mL-1, the values of MRT(0～∞h) were0.273h and0.383h in plasma and CSF respectively. The Tmax for catalpol and puerarin in CSF were0.083h and0.050h respectively. The AUCCsF/AUCplasma for catalpol and puerarin were7.2%and5.0%respectively. The results indicated catalpol and puerarin can be transported into BBB rapidly.In the CSF of the model, the distribution of catalpol and puerarin were significant higher and the eliminations of catalpol and puerarin were significantly slower than those observed in the normal, which may be related to the BBB disruption in the cerebral ischemia rats.3. Comparison the pharmacokinetics and biodistribution of catalpol and puerarin between the model and the normal ratsComparing with the normal rats, the Cmax and AUC(0～5h) of catalpol in the model rats plasma and were significantly lower than that in the normal (P<0.05), the Cmax of catalpol in the model was significantly higher than that in the normal (P<0.05), and the t1/2of puerarin in the model rats plasma was significantly longer than that in the normal (P<0.05), the Cmax and AUC(0～5h) of puerarin in the model rats CSF were significantly higher than that in the normal (P<0.05). The value of AUCCSF/AUCplasma was also significantly higher than that in the normal (P<0.05). The results indicated the biodistribution of catalpol and puerarin in the model rats CSF were high and suitable to the treatment of the cerebral ischemia.4. Investigate the distribution of puerarin in rat liver and kidneyA HPLC-UV method was developed and validated for the quantification of puerarin in liver and kidney to investigate the distribution of puerarin in rat liver and kidney after i.v. of Zige Freeze-dried Powder and Puerarin Freeze-dried Powder. The liver and kidney samples were pretreated with methanol for protein precipitation and the supernatants were separated on a Agilent C18column (250mm×4.6mm,5μm) with the mobile phase consisting of acetonitrile and water (15:85, v/v) at a flow rate of1.0mL·min-1. Detection was performed on HPLC with UV detector using peoniflorin as the internal standard (IS). The calibration curves had a good linear with r>0.999. The lower limit of quantification (LLOQ) was0.375μg·mL-1. The intra-and inter-day precision were found to be less than15%, and the accuracy were ranged in±15%for the biosamples. The developed and validated method was applied to research the distribution of puerarin in rats liver and kindey after i.v Zige Freeze-dried Powder and Puerarin Freeze-dried Powder.The results revealed that the contents of puerarin in kidney arrived the max values after10min of injection with58.1μg·g-1for the rats injected Zige Freeze-dried Powder and71.3μg·g-1for the rats injected Puerarin Freeze-dried Powder; the MRT(0-∞) was0.548h,0.564h for the Zige Freeze-dried Powder group and Puerarin Freeze-dried Powder group respectively. Compared with the rats injected Puerarin Freeze-dried Powder, the Cmax and AUC(0～2h) of that injected Zige Freeze-dried Powder was significantly lower (P<0.05) in kidney. Meanwhile, the contents of puerarin in liver arrived the max values of20.9μg·g-1,23.8μg·g-1, the AUC(0～2h) was9.5μg·h·g-1,10.1μg·h·g-1, and MRT(0-∞) was1.746h and1.455h.0.501h for the Zige Freeze-dried Powder group and Puerarin Freeze-dried Powder group respectively. Compared with the rats injected Puerarin Freeze-dried Powder group, there was no significant difference observed in liver. The results indicated that the Zige Freeze-dried Powder group reduced the accumulation of puerarin in rat kidney and would be more secure than the Puerarin Freeze-dried Powder group.5. Evaluating the ability and the mechanism of Zige Freeze-dried Powder in improvement of the permeability of BBBThe permeability of BBB to evans blue (EB) was investigated for evaluating the ability of Zige Freeze-dried Powder in improvement of the permeability of BBB, and then the distributions and expression of the tight junction (TJ) relative protein of Claudin-5, Occludin and ZO-1were studied for researching the potential mechanism of the improvement of the permeability of BBB.The results showed that Zige Freeze-dried Powder can decrease the permeability of EB to BBB. After7d, the contents of EB in the brain were2.06μg· g-1and0.97μg· g-1in the rats injected Zige Freeze-dried Powder and the model rats. It showed significantly low in the rats injected Zige Freeze-dried Powder compared with the model rats (P<0.05), which indicated the Zige Freeze-dried Powder could decreases the permeability of EB to BBB. After Zige Freeze-dried Powder i.v., the distributions and expression of Claudin-5, Occludin and ZO-1were increased, the significantly was observed after7d. The results indicated that the mechanism of Zige Freeze-dried Powder improving the BBB permeability of rats with MCAO may relate to increase the distributions and expression of Claudin-5, Occludin and ZO-1.Conclusions:Based on the results of the study, we can draw the conclusions:1) The pharmacokinetics of Zige Freeze-dried Powder and the distribution of catalpol and puerarin in CSF were studied in the normal and the model rats. The results will supply beneficial information to the clinical pharmacokinetic studies of Zige Freeze-dried Powder.2) In the model rats, the distribution of catalpol and puerarin in CSF were higher than that in the normal rat. It suggested that the Zige Freeze-dried Powder can be transported into BBB highly and is suitable to the treatment of the cerebral ischemic rats.3) The Zige Freeze-dried Powder could reduce the accumulation of puerarin in the rat kidney without effects on the elimination of puerarin in rat kidney, which indicates it may be more security than the Puerarin Freeze-dried Powder;4) The Zige Freeze-dried Powder could decrease the permeability of EB to BBB by improving the distributions and expression of Claudin-5, Occludin and ZO-1in the brain vessel. Overall, the study will be helpful in further explorations of the bioactive mechanism and clinical pharmacokinetics of Zige Freeze-dried Powder.
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