禁食和非禁食两种生理状态下大鼠口服绿原酸后血浆主要代谢物及生物利用度研究

Study of Plasma Major Metabolites and Bioavailability of Chlorogenic Acid after Oral Administration to Rats in Two Physiological States of Fasted and Non-Fasted

【目的】分析禁食(FT)和非禁食(NF)两种生理状态下大鼠口服绿原酸(CGA)后血浆主要代谢产物及其生物利用度特性。【方法】采用 UPLC-QTOF-MS 鉴定大鼠血浆中 CGA 的主要代谢产物;4 倍体积含 2% 乙酸的甲醇溶液沉淀血浆蛋白质,使用 ZORBAX RRHD EclipsePlus C18 柱(2.1 mm×150 mm,1.8 μm)作为固定相,以乙腈和 0.1% 甲酸水溶液作为流动相梯度洗脱,UPLC-QQQ-MS 在多反应监测模式下同时定量分析大鼠血浆中 CGA 及其主要代谢产物,使用非房室模型拟合药动学参数。【结果】FT 组主要代谢产物有 10 种:甲基绿原酸(CGA-Methyl)、绿原酸葡萄糖醛酸(CGA-Glu)、咖啡酸(CA)、咖啡酸葡萄糖醛酸(CA-Glu)、阿魏酸(FA)、阿魏酸葡萄糖醛酸(FA-Glu)、阿魏酸硫酸(FA-Sul)、二氢咖啡酸(DHCA)、奎宁酸(QA)和苯甲酸(BA),而 NF 组中未检测到 CA,只有 9 种;优化的检测方法用于 CGA 及其超出质谱检测限的代谢产物(DHCA、CGA-Methyl、CA-Glu 和 CGA-Glu)的生物利用度分析,口服 CGA 后,两种状态下大鼠血浆中 CGA 浓度均迅速上升,达峰时间(T_(max))为 0.5 h,大鼠血浆 CGA 的最大血药浓度(C_(max))、药时曲线下面积[AUC_((0-t))]随服用剂量的增加而增加,3个剂量 FT 组分别为(47.56±7.75),(118.21±12.18),(196.74±27.70)ng/mL 和(117.86±12.11),(315.05±44.18),(478.75±28.43)(ng·h)/mL,而 NF 组 C_(max)和 AUC_((0-t))均略低于 FT 大鼠,但 3个剂量 NF 组的半衰期(t_(1/2))比 FT 组分别延长了 1.21、0.65、0.41 h,平均驻留时间[MRT_((0-∞))]分别延长了 1.71,0.90,0.22 h。口服 CGA后,FT和 NF组的大鼠血浆中 CGA代谢产物的浓度均迅速上升,T_(max)在 0.5~2 h,除 DHCA含量在 NF组的略高于 FT组之外,其余代谢产物浓度变化的趋势与 CGA 基本一致。【结论】两种状态下 CGA 在大鼠血浆中代谢产物基本一致,除 NF组未检测到CA;正常饮食会降低CGA及其代谢物的吸收但会延长它们在体内驻留时间。

[Objective]The main plasma metabolites and their bioavailability properties after oral adminis-tration of chlorogenic acid(CGA)to rats in both fasted(FT)and non-fasted(NF)physiological states were ana-lyzed in this study[.Method]The major metabolites of CGA in rat plasma were identified by UPLC-QTOF-MS.A 4-fold volume of methanol solution containing 2% acetic acid was used to precipitate plasma proteins.A ZOR-BAX RRHD EclipsePlus C18 column(2.1 mm×150 mm,1.8 μm)was used as stationary phase,acetonitrile,0.1% formic acid aqueous solution was used as mobile phase for gradient elution,and UPLC-QQQ-MS for si-multaneous quantification of CGA and its major metabolites in rat plasma in multiple reaction monitoring mode,and finally pharmacokinetic parameters were fitted in a non-atrial model[.Result]There were 10 major metabo-lites in the FT group:methyl chlorogenic acid(CGA-Methyl),chlorogenic acid glucuronide(CGA-Glu),caffe-ic acid(CA),caffeic acid glucuronide(CA-Glu),ferulic acid(FA),ferulic acid glucuronide(FA-Glu),ferulicacid sulfate(FA-Sul),dihydrocaffeic acid(DHCA),quinic acid(QA)and benzoic acid(BA),while no CA wasdetected in the NF group and only 9 metabolites were present.The optimized detection method was used for bio-availability analysis of CGA and CGA metabolites(DHCA,CGA-Methyl,CA-Glu and CGA-Glu)that exceedthe detection limit of mass spectrometry.After oral administration of CGA,the concentration of CGA in rat plas-ma increased rapidly,and the time to peak(T_(max))was 0.5 h.The maximum blood concentration(C_(max)),area underthe drug-time curve(AUC_((0-t)))of rat plasma CGA increased with increasing dose,and the three dose FT groupswere(47.56±7.75),(118.21±12.18),(196.74±27.70) ng/mL and(117.86±12.11),(315.05±44.18),(478.75±28.43)(ng·h)/mL,respectively,The C_(max) and AUC_((0-t))of the NF group were slightly lower than thoseof the FT rats,but the half-lives(t_(1/2))of the three doses of NF group were prolonged compared with those of theFT group by 1.21,0.65,0.41 h,and the mean residence time(MRT(_(0-∞)))was prolonged by 1.71,0.90,0.22 h,re-spectively.The trends were consistent with those of CGA,except that DHCA was slightly higher in the NF groupthan in FT group[.Conclusion]The metabolites of CGA in rat plasma were essentially the same in both states,except that CA was not detected in the NF group;normal diet reduced the absorption of CGA and its metabo-lites,but normal diet prolonged their residence time in the body.