血小板胞内摄取莪术二酮的实验研究

Study on intracellular uptake of curdione in platelets

目的:通过对血小板胞内摄取莪术二酮的研究,为进一步阐明莪术中抗血小板活性成分的作用途径提供实验依据。方法:建立液相色谱-串联质谱(LC-MS/MS)方法检测血小板中莪术二酮的浓度并对其方法学进行验证;体外实验采用人洗涤血小板与不同浓度莪术二酮共培养,LC-MS/MS筛选血小板胞内摄取莪术二酮的最佳条件。体内实验通过采集莪术二酮溶液灌胃小鼠的血样和莪术油注射液滴注的临床患者血样,分步离心法提取血小板,LC-MS/MS检测血清与血小板胞内莪术二酮浓度。结果:莪术二酮质量浓度在1~200 ng·mL^(-1)范围内呈现良好线性(r=0.9975);定量限为0.4 ng·mL^(-1);方法的批内精密度与批间精密度均<5.0%,准确度均<8.8%;提取回收率在93.1%~110.2%。体外实验结果显示在血小板计数相同的条件下,莪术二酮(1、0.5、0.25 mmol·L^(-1))均能在血小板裂解液中检测到;体内实验结果显示灌胃小鼠和给药患者的血小板裂解液、血清中均检测有莪术二酮;实验证明莪术二酮在体内外均可被血小板结合和摄取。结论:血小板可以胞内摄取莪术二酮,为进一步研究莪术二酮抗血小板作用机制提供实验基础。

Objective:To provide experimental basis and further clarify the pathway of anti-platelet active components of Curcuma zedoaria by the study on the intracellular uptake of curdione by platelets.Methods:Establishment of a liquid chromatography-tandem mass spectrometry(LC-MS/MS)method to detect the concentration of curdione in platelets and validation of the methodology.In vitro,the washed human platelets were co-cultured with different concentrations of curdione.LC-MS/MS was used to screen the optimal conditions for the uptake of curdione.In vivo blood samples were collected from mice orally administered with curdione and clinical patientsdosed with Zedoary Turmeric oil injection.Platelets were extracted by stepwise centrifugation.Afterwards,LC-MS/MS was performed to detect intracellular curcumin concentrations in serum and platelets.Results:The results indicated that curdione in platelets had a good linear relationship in a range of 1-200 ng·mL^(-1)(r=0.9975).The limit of detection was 0.5 ng·mL^(-1).The intra-and inter-batch precision were less than 5.0%,the accuracy were less than 8.8%and the average recoveries were 93.1%-110.2%.The results in vitro showed that curdione(1,0.5,0.25 mmol·L^(-1))could be detected in platelet lysates under the same platelet count condition.In vivo,the data showed that curdione was detected in platelet of mice and patients.These results indicated that curdione could be uptaken by platelets in vitro and in vivo.Conclusion:Platelets can intracellularly uptake curdione.It provides experimental basis for further study on the mechanism of antiplatelet effects of curdione.