| 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 623â†'161 and m/z 289â†'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. |
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