血栓素B2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TXB₂含量的方法.血小板TXB₂用Sep-PakC₁₈提取,在冠醚和碳酸钾存在下,BrMmc与TXB₂的羧基反应生成BrMmc-TXB₂。然后经Zorbax-ODS柱(250mm×4.6mm,5μm)分离,测定荧光强度(E×345,Em405)。流动相为乙腈:水:磷酸(60:40:0.1V/V),流速1ml/min.BrMmc-TXB₂的分离在20min内完成。TXB₂的含量用外标法定量,检测限15ng。体外实验刺五加提取物(0.275～2.2mg/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TXB₂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 TXB2 Content in Platelet and the Anti-platelet Actions of Acathopanax Senticosus

TXB₂ 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 TXB₂ were extracted by a Sep-Pak C₁₈ (waters).In the presence of 18-crown-6 and K₂CO₃,BrMmc reacted with carboxylic group(—COOH) of TXB₂ to form a fluorescent ester (BrMmc-TXB₂),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-TXB₂ was completed in 20min.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 TXB₂ 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.