The Seven-party Anti-platelet Aggregation. Dan Proteomics And Pharmacokinetics Studies

(1)Salvia miltiorrhiza and Panax notoginseng were both considered to be beneficial to cardiovascular diseases in traditional Chinese medicine and often used in the form of combination.To check the possible interaction between them,the effects of the active components of these two plants,salvianolic acids(SA)and Panax notoginseng saponins(PNS), on platelet aggregation were checked separately or as combination in vitro and ex vivo.In vitro study indicated that both SA and PNS have inhibitory effect on platelet aggregation,including platelet rich plasma(PRP)and washed platelet after ADP induction.But,the combination of SA and PNS showed no synergistic effect in vitro.I.g.550 mg·kg^-1·d^-1of SA or PNS for 5 days could significantly inhibit ADP-induced platelet aggregation in PRP.Combination of SA and PNS at ratio of 5:1 exhibited synergistic effect on platelet aggregation of PRP.We also studied salvianolic acid B(SalB),a purified compound from Salvia miltiorrhiza,which showed that it could inhibit the ADP-induced aggregation of rat washed platelets with an IC₅₀-value of 67.33±1.22μg·mL^-1in vitro.(2)To elucidate the cellular mechanism of the inhibitive effect on platelet aggregation,the protein expression profiles of rat platelets with or without treatment of SA or PNS or SalB were checked using 2-DE and the proteins altered in expressional level were identified by MALDI-TOF-MS.The treatment of SA or PNS or SalB caused the regulation of expression levels of several proteins,which play important roles in calcium homeostasis,platelet activation, transmembrane signaling,energy metabolism,antioxidant system,cytoskeleton structure and etc. The regulation of proteins was also confirmed by Western blotting.(3)A high-performance liquid chromatography(HPLC)method was established for the simultaneous determination of four phenolic acids(procatechualdehyde,rosmarinic acid, lithospermic acid,salvianolic acid B)in rat plasma after oral administration of salvianolic acids and Panax notoginseng saponins(5:1),and studied its pharmacokinetics.Chromatographic separation was achieved on a Zorbax SB-C₁₈column(250 mm×4.6 mm,5μm)at 30℃,with a linear gradient elution of acetonitrile and 0.026%aqueous phosphoricacid(v/v),at a flow rate of 1 mL·min^-1.The eluent was detected at 280 nm for the four phenolic acids and internal standard. The plasma sample was extracted with ethyl acetate twice after acidification by 10% hydrochloric acid,then calculated the key pharmacokinetic parameters.The study indicated that the within- and between-day assay coefficients of variation for the four phenolic acids in plasma were less than 15%and the extraction recovery was higher than 70%.The pharmacokinetic parameters for the four phenolic acids were as followes:T_max,C_maxand AUC_0-240for procatechualdehyde were 10.0 min,6.466μg·mL^-1and 82.0μg·min·mL^-1,respectively;for rosmafinic acid were 11.7 min,10.50μg·mL^-1and 446.6μg·min·mL^-1,respectively;for lithospermic acid were 20.8 min,5.950μg·mL^-1and 486.7μg·min·mL^-1,respectively;for salvianolic acid B were 8.33 min,48.12μg·mL^-1and 1766μg·min·mL^-1.(4)After oral administration of salvianolic acids and Panax notoginseng saponins(5:1)in rat at 5～240min,the concentrations of four phenolic acids(procatechualdehyde,rosmafinic acid, lithospermic acid,salvianolic acid B)in some tissues were analyzed by HPLC-UV method,and the features of distribution were evaluated.The study indicated that only procatechualdehyde could be found in liver,kidney,lung and spleen.Procatechualdehyde was quickly absorbed and distributed into these tissues,the highest levels were attainedat 10～30min,and then declined rapidly.But in lung the concentration-time curve of procatechualdehyde was the double peaks. The drug concentration in tissues were lower than that in blood at the same time point obviously.