| Coleus forskohlii (Willd) Brig belongs to the Labiatae family. Its dried herb, which is called Qiaoruisu, is recorded in Quality Standard of the Native Yunnan Herb (2005Edition) and applied in clinic for the treatment of cold, cough and asthma, etc. C. forskohlii contains abundant labdane-diterpenes, of which isoforskolin is the main active component in native species. It has been reported that isoforskolin exerts significant antispasmodic, blood pressure lowering and ocular hypotensive effects. However, existing pharmacological studies of isoforskolin usually focus on in vitro animal model and there's still no data obtained from whole animal models. Besides, no pharmacokinetic parameters of isoforskolin are available so far. Therefore, the present study mainly emphasized on the antiasthmatic effect of isoforskolin on histamine-induced asthma guinea pig and the pharmacokinetics of isoforskolin.1. Isolation and purification of isoforskolin from C. forskohliiDried herb of C. forskohlii (8kg) was powdered, immersed, and percolated with80%ethanol at room temperature. The percolate was evaporated in vacuum (?60?) to give1.2kg of extract. Repeated column chromatography and semi-prepared RP-HPLC of the extract afforded690mg of isoforskolin. The purity of isoforskolin was assessed by HPLC-ELSD and calculated by peak area normalization method as?98%.2. Antiasthmatic effect of isoforskolinThe effect of isoforskolin on histamine-induced spasm of isolated guinea pig tracheas was explored by adding successive dose of isoforskolin onto the tracheal strips. Isoforskolin (0.5-20?g/mL) produced a dose-dependent relaxation effect on isolated trachea strips of guinea pigs and the effect reached a platform at the concentration of10?g/mL. Compared with blank control group and positive drug aminophylline group, isoforskolin group showed significant antiasthmatic action in vitro.The investigation of antiasthmatic effect of isoforskolin on histamine-induced asthma guinea pigs in vivo showed that isoforskolin could significantly prolong the incubation period of expiratory dyspnea at three doses of0.75,1and1.25mg/kg. All three time intervals (30,60and90min) between isoforskolin administration (i.p.) and trachea spasm inducement at the dose of0.75mg/kg could significantly prolong the incubation period of expiratory dyspnea, of which an interval of60min was the best.The studies above show that isoforskolin has significant antiasthmatic effect both on isolated trachea strips of guinea pigs in vitro and on histamine-induced asthma guinea pigs in vivo.3. Pharmacokinetic study of isoforskolin in beagle dogsA sensitive and specific high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method has been developed and validated for the determination of isoforskolin in canine plasma. The chromatographic separation was performed on a Venusil XBP-PH C18column with an isocratic mobile phase consisting of methanol-2mM ammonium acetate-formic acid (62:38:0.1, v/v/v), pumped at0.35mL/min. Isoforskolin and the internal standard (I.S.) eplerenone were detected at m/z433.4?373.3and m/z415.3?163.5in multiple reaction monitoring (MRM) positive mode, respectively. The standard curves were linear over the concentration range of0.1-200ng/mL, with r>0.995. The intra-and inter-batch accuracy for isoforskolin at four concentrations was90.2-108.3%and97.8-106.6%, respectively. The RSDs were less than6.0%. The average extraction recoveries of isoforskolin and I.S. were97.0and88.4%, respectively. The method was successfully applied to the pharmacokinetic study after a single intravenous administration of isoforskolin (1.0mg/kg) in beagle dogs. The concentration-time profiles were fitted a two-compartmental model. Key pharmacokinetic parameters were as follows:t1/2?1.50h and AUC0-t326.5±90.1ng h/mL.In the end, a review on the application of new analytical techniques in the pharmacokinetics of traditional Chinese medicine is presented. |
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