| Guan xin danshen formula is a classic prescription in2005edition ofpharmacopoeia, which includes salvia miltiorrhiza, notoginseng anddalbergiae. The main pharmacological function is to improve the bloodsupply, protect myocardial ischemia and vascular endothelial cells, improvelipid metabolism and hinder the formation of thrombus. Besides, it canprotect cerebral ischemia to some degree. A large number of researchesshow that it plays an impotent role in protection of the cerebral ischemia.And there is less pharmacokinetic research on it.A effective and sensitive LC-MS/MS method was developed in rat’sbrain and plasma by using protocatechuic acid, protocatechuic aldehyde,salvianolic acid A, salvianolic acid B, cryptotanshinone and TanshinoneⅡA. SD rats were subjects, and the brain micro-dialysis and automatic blooddialysis technology were combined, and then micro-dialysis probe waslocated in rat brain ventricles, and automatic blood meter connected to thejugular vein, and brain micro-dialysis and blood samples were collected.Screen the ingredients that can cross the blood-brain barrier, and map theirconcentration time curve. And pharmacokinetic software was used tocalculate the main pharmacokinetic parameters and log BB was calculatedto compare the ratios of brain blood distribution at different times. Furtherstudy on the brain and plasma pharmacokinetic characteristics was carriedout, and then its ability to cross the blood-brain barrier was compared. Therats were iv. administration of PCA, PAL, SAA, SAB, CTS and TSⅡA,and the doses were25、20、4、15、10、25mg·kg-1. After iv. administrationof PCA, PAL, SAA, SAB, CTS and TSⅡA at a dose of20mg·kg-1, the micro-dialysis samples were collected at10,20,30,40,50,60,70,80,90min, and the blood samples were collected at5,10,20,30,40,50,60,70,80,90min. The samples were separated on an Agilent Eclipse Plus-C18column (2.1×50mm,3.5μm) in gradient elution mode. The massspectrometer was operated under both positive and negative ion mode withthe ESI source, and the detection was performed by MRM. In positivemode the transition of297.4/254.3m/z for cryptotanshinone,295.5/249.3m/z for tanshinoneⅡA and285.2/154.0m/z for Diazepam. In negative ionmode the transition of154.3/153.1m/z for protocatechuic acid,137.3/108m/z for protocatechuic aldehyde,493.0/295.2m/z for Salvianolic acid A,718.0/520.0m/z for Salvianolic acid B,321.4/152.3m/z forChloramphenicol. The plasma and brain samples were precipitated withethyl acetate, and the micro-dialysis samples were detected after addedinternal standard. Then detect the calibration and use the probeyieldcorrection to calculate the real drug concentration. The calibration curves inthe range of1.25~1000ng·mL-1for cryptotanshinone, and the LLOQ is1.25ng·mL-1;0.625~1000ng·mL-1for TanshinoneⅡA, Protocatechuic acidand protocatechuic aldehyde, and the LLOQ is0.625ng·mL-1;1.25~1000ng·mL-1for Salvianolic acid A, and the LLOQ is1.25ng·mL-1;2.5~1000ng·mL-1for Salvianolic acid B, and the LLOQ is2.5ng·mL-1. They all withgood linearity in rat plasma and brain. The analysis method is sensitive,simple and suitable enough to be applied in the pharmacokinetic study ofthe6ingredients. Animal testing gives the'BB of the drugs, CTS>TSⅡA> PAL> PAC>SAA≈SAB when at40min, and TSⅡA> CTS> PAC>PAL≈SAA≈SAB at90min. Further studies of the6ingredients in ratsturn out that PAC, PAL, SAA, CTS, TSⅡA can cross the blood-brainbarrier, while SAB can not. Using the software of WinNonlin tocalculate the main pharmacokinetic parameters, and study theirpharmacokinetics.In summary, we select6kinds of effective ingredients of Salvia miltiorrhiza, then the animal experiments were taken by brain micro-dialysis, automatic blood sampler and traditional brain homogenate method,the samples were analyzed by LC-MS/MS. Then we selected theingredients which can across the blood-brain barrier and described theirdynamics characteristic in the brain and plasma. |
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