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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。
In this paper, based on the fluorescence derivatization experiments of Turk and Wintersteiger, a method was established for the determination of TXB_2 in platelets by reverse-phase HPLC.The platelet TXB_2 was extracted with Sep-Pak C_ (18), and in the presence of crown ether and potassium carbonate, BrMmc and TXB_2 Carboxyl reaction to generate BrMmc-TXB_2. The fluorescence intensity (E × 345, Em 405) was then determined by separation on a Zorbax-ODS column (250 mm × 4.6 mm, 5 μm). The mobile phase was acetonitrile: water: phosphoric acid (60:40: 0.1 V / V) at a flow rate of 1 ml / min. The separation of Brmmc-TXB_2 was completed within 20 min. The content of TXB_2 was determined by external standard method with the detection limit of 15 ng. In vitro experiments Acanthopanax senticosus extract (0.275 ~ 2.2 mg / mlPRP) AA, ADP induced rabbit platelet aggregation was significantly inhibited, and can inhibit AA-induced platelet TXB_2 generation. Intravenous injection (120 mg / kg) also inhibited AA and ADP-induced aggregation.