连翘酯苷的吸收及代谢研究

【作者】 王庚南；

【导师】 常琪；

【作者基本信息】 中国协和医科大学 ， 生药学， 2010， 硕士

【摘要】 连翘为木犀科植物连翘(Forsythia suspens (Thunb.) Vahl)的干燥果实。味苦性寒,具有清热解毒的功效,尤其对有病毒或细菌引起的上呼吸道感染有显著地疗效。连翘中所含苯乙醇类成分具有保护肝脏、消退黄疸的功效,其苷元咖啡酸及结构类似物绿原酸也均有护肝利胆作用。连翘酯苷(forsythiaside, FTA)属于苯乙醇苷类且是连翘的主要有效成分之一。FTA和连翘苷通常被作为连翘药材的主要质控及制备成分。文献报道,FTA在青翘和老翘中的含量分别4-7%和0.8-3%。近年来,对FTA的药理作用的研究中,发现其具有抗菌、抗病毒、抗氧化以及松弛血管等生物活性,但对FTA的药代动力学研究甚少。本论文采用高灵敏度的LC-MS/MS技术,以整体实验动物和体外细胞模型研究给予连翘酯苷后的药代动力学特性,包括其原型药物成分的吸收及代谢产物的测定。阐释FTA的药代动力学过程和规律。首先,本文首次建立了高效、灵敏、稳定可靠地LC-MS/MS分析方法测定FTA在大鼠血浆中的含量。本方法选用表儿茶精作为内标物,并采用固相萃取法作为样品前处理方法。结果显示,FTA在大鼠血浆中的浓度在2.0～50.0和50.0～5000.0ng/mL范围内呈现良好的线性,并且此方法的最低检测限和最低定量限分别为0.2和1 ng／mL。同时呈现出了良好的精密度(RSD<10.8)和准确度>91.9%,且提取回收率在81.3%～85.0%之间,均达到生物样品测定方法学的要求。其次,本文首次应用已经建立的LC-MS／MS分析方法,研究了FTA在大鼠体内的药代动力学特性。体内药代动力学结果表明大鼠口服FTA(100 mg／kg)后,达峰时间非常短(Tmax,20min),但其最大血药浓度低(Cmax,122.2±45.4 ng／mL),并且在体内消除快,静脉和口服FTA后其t1／2,λz分别为76.8±26.5 min及74.7±13.3 min,其AUCo-t分别为570.5±69.2μg min／mL及13.9±5.2μg min／mL。研究表明FTA的生物利用度很低,仅为0.5%。FTA机体稳定性的研究结果显示大鼠胃内容物对FTA无明显影响,而在小肠和大肠内容物中FTA均有不同程度的降解,其中在大肠内容物中降解最为明显,其降解率为38.7%。并且在FTApH环境稳定性研究中发现FTA在酸性下稳定,而在弱碱性和碱性条件易降解,从而表明在血浆和大肠中pH环境是引起FTA稳定性下降的主要因素。再次,本文首次建立了体外Caco-2细胞模型,并以此模型对FTA在人体肠壁吸收机理进行研究,结果显示：FTA透膜能力差,其表观渗透系数(Papp)仅为1.2×10-7cm／s,属于吸收不良的药物,在Caco-2细胞模型上,不同浓度的FTA从AP→BL面和BL→AP面的Papp值无统计学差异,且其Papp值不会受药物浓度的增加而改变而转运量却会随浓度的增加而增加。综上所示,FTA在Caco-2单层细胞模型上,主要的转运方式为被动细胞转运。第三,本文首次对FTA的排泄途径进行了研究。研究结果显示,在静脉给予大鼠FTA(20 mg／kg)后,经过11 h,尿液中累积排泄率为给药量的12.7±4.6%,而在胆汁中经过3 h的累积排泄率为给药量的1.36±0.31%。结果表明,FTA从胆汁和尿液中原型的排泄量约占给药量的15%,而其余药物可能转化为代谢物形式排出。最后,本文首次研究了FTA的代谢途径,结果显示FTA在胆汁中拥有较强的代谢,利用LC-MS／MS中TIC模式和Product Ion模式鉴定出二相代谢物10种经LC-MS分析,与空白样品对比,在静脉给药后大鼠的尿样和胆汁中找到了FTA原型及其10种代谢物,分别为FTA单甲基化产物M1(m/z 637)；FTA双甲基化产物M2(m／z651)；FTA甲基化和硫酸化代谢物M3、M4(m／z 717、731)以及M3、M4的还原产物M5、M6(m／z 719、733)；FTA甲基化和葡萄糖醛酸化产物M7、M8(m／z 813、827)以及M7、M8的还原产物M9、M10(m／z 815、829)。本论文研究结果系统的揭示了FTA吸收代谢规律,为苯乙醇苷类化合物的研究提供了实验依据,有利于正确评价此类药物的安全性与有效性,为此类药物的开发利用提供参考。更多还原

【Abstract】 Forsythiaside (FTA), a phenylethanoid glycoside (Fig.1), is the most abundant component of a very well-known Chinese herbal medicine Lian-Qiao, which is the fruit of Forsythia suspense (Thunb) Vahl. The herb has been widely used as an antipyretic, antidotal and anti-inflammatory agent in China, Japan and Korea for the treatment of various infections, especially acute upper respiratory tract complaints caused by viruses and/or bacteria infection.FTA together with phillyrin are commonly used as chemical markers for quality control of Lian-Qiao raw material and the derivated preparations . The content of forsythiaside is about 4-7% in its dry green fruits named Qing-Qiao and 0.8-3%in its ripe fruits named Lao-Qiao Pharmacological studies demonstrated that FTA possesses strong antioxidant , antibacterial and antiviral activities, and also exhibits a slow relaxation effect against norepinephrine induced contraction of rat aorta.Moreover, it is reported that FTA could significantly protect DNA damage caused by hydroxyl radicals and inhibit protein kinase C (PKCa) with an IC50 value of 1.9μM. However, the pharmacokinetic characteristics and absorption profile in the gastrointestinal tract of FTA are largely unknown so far, due to lack of sensitive assays.Firstly, a highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of FTA in rat plasma using epicatechin as internal standard. The analytes were extracted by solid-phase extraction and chromatographied on a C18 column eluted with a gradient mobile phase of acetonitrile and water both containing 0.2% formic acid. The detection was performed by negative ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 623→161 and m/z 289→109 for FTA and I.S., respectively. The assay was linear over the concentration ranges of 2.0-50.0 and 50.0-5000.0 ng/mL with limits of detection and quantification of 0.2 and 1.0 ng/mL, respectively. The precision was <10.8%and the accuracy was>91.9%, and extraction recovery ranged from 81.3%to 85.0%.Secondly, a highly selective LC-MS/MS method has developed for quantificaiton of FTA in rat plasma to understand the pharmacokinetics and oral bioavailability of FTA in rats. And the results presented the absoption of FTA was extremely fast in rats (Tmax, 20 min) after intragastric admimistratiaon (100 mg/kg). However, the plasma concentration maximum was very low (Cmax,122.2±45.4 ng/mL). The elimination was fast after both intraveneous and intragastric administration (t1/2,λz 76.8±26.5 and 74.7±13.3 min). And the AUC0-t for intraveneous and intragastric administration were 570.5±69.2 and 13.9±5.2μg min/mL. All the pharmacokinetic parameters demonstrated the bioavailability of FTA in rat was very low in this study, just about 0.5%.Thirdly, stability of FTA by peptidases was investigated by in cubation of the drug with different pH condition, the contents of stomach, small intestine, colon and plasma at 37℃.It showed that at pH 1.2 and pH 6.8, FTA was stable with no significant degradation for which is unstable at pH 7.4. Furthermore, FTA was stable in the stomach while unstable in the small intestine and colon. And after 4h incubation with their contents, FTA were degradated with 21%decrease in small intestine and 39%in colon, respectively. Compared with the two results, it presented that the degradation caused by the chemical (pH) instabilities in colon and plasma can not be ignored. Moreover the bacteria or enzymes in small intestine and colon played an important role in the degradation of FTA.Moreover, In the Caco-2 cell model, the absorption transport of FTA was fairly poor with Papp values of 1.2×10-7cm/s. The bidirectional studies were performed under non-gradient conditions at a concentration of 100-300μM. Transport of FTA was not changed by the different concentrations. So, transport route of FTA was the passive transport.Finally, the metabolites of FTA in urine and bile was determined by the LC-MS/MS. The intact form of FTA in the body was mainly excreted from urine at 12.7±4.6%of dose, and slightly excreted from bile at 1.4±0.3%of dose. FTA was extensively transferred into its methylated, sulfated or/and glucuronidated metabolites and then excreted from both urine and bile. The main metabolites of forsythiaside were characterized as its monomethylated metabolite Ml at m/z 637; bimethylated metabolite M2 at m/z 651; both methylated and sulfated metabolites M3 and M4 at m/z 717,731 and their reduced products M5 and M6 at m/z 719,733; both methylated and glucuronidated metabolites M7 and M8 at m/z 813,827 and their reduced products M9 and M10 at m/z 815,829.更多还原