血栓素B_2的高效液相荧光测定及刺五加提取物的抗血小板作用

本文在Turk 及 Wintersteiger 荧光衍生实验的基础上,建立了反相 HPLC 法测定血小板 TXB_2含量的方法.血小板 TXB_2用 Sep-Pak C_(18)提取,在冠醚和碳酸钾存在下,BrMmc 与 TXB_2的羧基反应生成 BrMmc-TXB_2。然后经 Zorbax-ODS 柱(250mm×4.6mm,5μm)分离,测定荧光强度(E×345,Em 405)。流动相为乙腈:水:磷酸(60:40:0.1V/V),流速1 ml/min.BrMmc-TXB_2的分离在20 min内完成。TXB_2的含量用外标法定量,检测限15ng。体外实验刺五加提取物(0.275～2.2 mg/mlPRP)对 AA、ADP 诱导的家兔血小板聚集有明显的抑制作用,并能抑制 AA 诱导的血小板 TXB_2的生成。静脉注射(120mg/kg)对 AA、ADP 诱导的聚集也有抑制作用。

HPLC Analysis of TXB_2 Content in Platelet and the Anti-platelet Actions of Acathopanax Senticosus

TXB_2 content in platelet were analysed by high-performance liquid chromatography (HPLC) using a pre-column derivatization method,in which a fluorescentreagent,4-bro- momethyl-7-methoxycoumarin (BrMmc) was used.The mothod was applied to studing the effects of Acanthopanax Senticosus (A.S.) extract,a Chinese medicine,on platelet func- tion.The platelet TXB_2 were extracted by a Sep-Pak C_(18) (waters).In the presence of 18- crown-6 and K_2CO_3,BrMmc reacted with carboxylic group (—COOH) of TXB_2 to form a fluorescent ester (BrMmc-TXB_2),which was seperated on a column (250mm×4.6mm I.D.)packed with Zorbax-ODS (6 μm,Dupont Instruments)and monitored by a fluorescent detector in Ex 345 nm and Em 405 nm.The mobile phase (acetonitrile:water:phosphoric acid;60:40:0.1 V/V),flowed at 1 ml/min at 30℃.The seperation of BrMmc-TXB_2 was completed in 20 min.The detection limit was 15 ng using the external standard method.The anti-platelet actions of A.S.in rabbit were studied.The resultes (in vitro) showed that the platelet aggregations induced by AA and ADP were potently inhibited by A.S.in a dose-de- pendent manner and the AA-induced TXB_2 formation in platelet was also reduced.After in- travenous injection of A.S.(120mg/kg),the platelet aggregations induced by AA and ADP were inhibited and the effects lasted 30 and 45 min,respectively.