| Anemarrhenae Rhizoma is a dried rhizome of Liliaceae plant,and it’s also a famous traditional Chinese herbal medicine that has been included in the《Chinese Pharmacopoeia》which has the functions of clearing heat and purging fire,nourishing yin and moisturizing dryness.Zhimu mainly contains a series of saponins and flavonoids.Modern pharmacological studies have shown that it has anti-tumor,anti-inflammatory,antipyretic,anti-diabetic,anti-depression,anti-platelet aggregation and anti-aging,improve learning and memory activities.Zhimu and its extracts can significantly improve the learning and memory ability of AD model animals,and have the inhibitory activity of cholinesterase related to the treatment of AD;in addition,it also reduces the deposition of A?by enhancing the function of cholinergic neurons,ameliorate memory defects caused by long-term cholinergic degeneration.Therefore,Zhimu often appears in clinical prescriptions for the treatment of AD.However,the mechanism of Zhimu treatment of AD is not yet clear,and the transport of its active ingredients across the organ barrier is still unknown.Therefore,this article has conducted the following three aspects of research on various active ingredients of Zhimu:1.Research on multi-components transmembrane transport of Zhimu based on in vitro Caco-2 cell modelObjectives:To explore the transport mechanism of various components of Zhimu across intestinal,and to elucidate the relationship between the components of Zhimu and transport proteins.Methods:Caco-2 cells were used to establish the intestinal absorption model of monolayer cells,and its structure and function were evaluated.The transmembrane transport experiment was carried out with the appropriate concentration of Zhimu multi-component that timosaponins AIII,timosaponins BII,mangiferin,isomangiferin,neomangiferin,baoguoside I and sarsasapogenin.The content of the above components in the cell transport samples was determined by the established HPLC/QQQ/MS quantitative method,so as to analyze the transport of the target components across the intestinal absorption barrier.Results:Caco-2 cells were used to establish an intestinal absorption barrier cell model with a TEER of 552Ω·cm2 and a sodium fluorescein permeability coefficient of（0.28±0.04）×10-6 cm/s;P-gp inhibition of ketoconazole can inhibit the efflux of rhodamine 123 and increases the absorption of rhodamine 123 in this model.The established HPLC-QQQ/MS detection method has good specificity,accuracy and precision all meet the requirements;the transmembrane transport results show that the Papp（AP-BL）of mangiferin,isomangiferin,neomangiferin,baohuoside I,timosaponin AIII,timosaponin BII and and sarsasapogenin are（8.524±1.706）×10-8,（40.758±5.872）×10-8,（102.807±17.238）×10-8,（18.884±1.587）×10-8,（44.087±1.413）×10-8,（10.041±0.791）×10-8 and（32.018±4.633）×10-8cm/s,the efflux rate（ER）of isomangiferin,baohuoside I,timosaponin BII,and sarsasapogenin are greater than 2,the efflux rate of the 7 active components decreased significantly after being combined with ketoconazole.Conclusion:The active ingredients of Chinese medicine Zhimu,isomangiferin,baohuoside I,timosaponins BII and sarsasapogenin are P-glycoprotein substrates.The absorption of mangiferin,neomangiferin,and timosaponins AIII on the Caco-2 cell model may be a purely passive transcellular transport,in which the neomangiferin is well absorbed and is not affected by P-gp inhibitors.2.Study on the pharmacokinetics of multi-components of Zhimu extracts in rats and the effect of P-glycoprotein on itObjectives:To explore the pharmacokinetic characteristics of Zhimu multiple components in animals and the effect of P-glycoprotein on the in vivo processes of each component.Methods:Establish the HPLC-QQQ/MS quantitative method for the multiple components of siblings in plasma,process plasma samples by protein precipitation method,gradient elution,flow rate is 0.300 m L/min,mass spectrometry multiple reaction monitoring mode perform quantitative analysis.Sixteen rats were randomly divided into a control group and an inhibitor group.The rats in the inhibitor group were orally administered ketoconazole solution（10 mg/kg,1 m L/200 g）,and the rats in the control group were orally administered normal saline（1 m L/200 g）.After30 minutes,all rats were given gavage extracts of Zhimu（60 g/kg,2 m L/200 g）.After intragastric administration of Zhimu extracts 0.25 h,0.5 h,1 h,1.5 h,2 h,3 h,4 h,6 h,8 h,12 h,24 h,30 h,36 h,blood samples were collected from 8 rats in control group and 8 rats in inhibitor group according to the administration sequence.The pharmacokinetic parameters were fitted by measuring the concentration of various components of Zhimu in plasma samples,and the effect of P-gp inhibitor ketoconazole on the process of Zhimu components in vivo was evaluated.Results:The specificity of the seven components of HPLC-QQQ/MS method in plasma was good,the intra-day and inter-day precision RSD were less than 11%,the matrix effect was 87.06-123.85%,the stability met the requirements;6 other components except for baohuoside I were detected in plasma samples of the control group.Compared with the control group,all 7 components including baohuoside I were detected in the plasma of the inhibitor group;mangiferin and isomangiferin were well absorbed in rats with higher AUC and Cmax values;sarsasapogenin had the longest half-life and elimination time among the 7 components,and its Tmaxvalue was significantly extended after the addition of the p-glycoprotein inhibitor ketoconazole.Conclusion:Mangiferin,isomangiferin and neomangiferin were absorbed rapidly in rats;timosaponin BII was well absorbed and was a P-glycoprotein substrate;timosaponins AIII was generally absorbed but eliminated for a long time in rats;baohuoside I is a P-glycoprotein substrate and the bioavailability of oral administration is extremely low;the trans-intestinal barrier of sarsasapogenin is affected by P-glycoprotein,which may be a P-glycoprotein substrate.3.Study on the distribution of multi-components of Zhimu extract in rat brainObjectives:To explore the distribution of the components of Zhimu in the animal across the blood-brain barrier to the brain.Methods:After giving normal SD rats a dose of 60 g/kg（2 m L/200g）by gavage the extracts of Zhimu,at 1 h,3 h,6 h,9 h,and24 h,the concentrations of Zhimu multiple components in plasma and brain tissue of rats were measured,and the translocation of Zhimu multicomponent across BBB was analyzed.Results:The HPLC-QQQ/MS quantitative method of 7 components of Anemarrhenae Rhizoma in brain tissue was established,and the specificity,precision and accuracy,matrix effect and stability met the requirements.Among them,neomangiferin,timosaponin BII,timosaponin AIII and sarsasapogenin can be transported across the BBB to the brain,while the amount of mangiferin and isomangiferin transported across the BBB to the brain tissue are very small;meanwhile,the baohuoside I can not be detected in blood,nor can be transported across the BBB to brain tissue.The ratio of neomangiferin,timosaponin BII,and sarsasapogenin keeps increasing at different time points,neomangiferin and sarsasapogenin are quickly absorbed into the brain via blood,at the same time,the brain-plasma ratio of sarsasapogenin reached 147%at 24 h,indicating that sarsasapogenin can be transported across the BBB barrier to the brain and was well absorbed,while the brain-plasma ratio of timosaponin BII was small at 9 h and increased nearly tenfold at 24 h,indicating that its drug metabolism in the brain is significantly slower than that in the blood,presumably due to the effect of P-glycoprotein efflux,There was no significant change in the brain-plasma ratio of timosaponin AIII at different time points,indicating that its elimination rate in brain and blood was basically the same.Conclusion:Neomangiferin,timosaponin BII,timosaponin AIII and sarsasapogenin can be transported across the BBB to the brain.Among them,neomangiferin and sarsasapogenin are absorbed faster in the brain,and the elimination rate of sarsasapogenin in the brain is significantly lower than that in the blood,and the transport of timosaponin BII across the BBB to the brain is greatly affected by the efflux of P-glycoprotein.In summary,the isomangiferin,baohuoside I,timosaponin BII and sarsasapogenin in Zhimu may be P-glycoprotein substrates;mangiferin and isomangiferin can be quickly absorbed into the blood after oral administration which had good bioavailability;neomangiferin,timosaponin AIII,timosaponin BII,and sarsasapogenin can be transported across the BBB to the brain,of which neomangiferin and sarsasapogenin are absorbed into the brain faster,sarsasapogenin has the largest brain-plasma ratio and the longest elimination time in blood and brain,which is speculated to be the main effective ingredient in the brain to prevent and cure Alzheimer’s disease. |
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