| Objective: An analysis method was to be developed for the quantification of rosamultin and kaji-ichigoside F1 in Potentilla anserina L., which was selected as the target traditional medicine. And the preliminary pharmacokinetics of rosamultin in rats was to be explored for the further research of its medicine value.Methods:1. The 30% ethanol solution of alcohol extract of Potentilla anserina L. was loaded on D101 macroporous resin and gradiently eluted with water, 30% Et OH, 70% Et OH, 95% Et OH. The 70% Et OH fraction was then chromatographed on silica gel, ODS and preparative RP-HPLC to isolate rosamultin for the accumulation material needed in pharmacokinetic study.2. An RP-HPLC-ELSD method was developed and validated for the determination of rosamultin and kaji-ichigoside F1 in Potentilla anserina L.. Using the method to determine the two components in 12 different Potentilla anserina L. samples available from Xining Qinghai, Yushu Qinghai, Dali Yunnan, Chuxiong Yunnan, Naqu Tibet, Kangding Sichuan and Gannan Gansu.3. An SPE-RP-HPLC-MS method was developed and validated for the determination of rosamultin in rat plasma. Using this method to determine the drug – time profile in rats after oral administration of rosamultin, and preliminary pharmacokinetic parameters was calculated.Results:1. In the present study, 4 triterpenoids were rapidly isolated from alcohol extract of Potentilla anserina L. by D101 macroporous resin, open column chromatography, ODS and preparative RP-HPLC.2. The determination of rosamultin and kaji-ichigoside F1 in 12 different Potentilla anserina L. samples were carried out using an HPLC-ELSD method. Linear calibration(r2=0.9981 for rosamultin, r2=0.9992 for kaji-ichigoside F1) curves were obtained over the concentration range of 34.80~754.00 ?g/ml for rosamultin and 34.23~741.65 ?g/ml for kaji-ichigoside F1. Precision(intra- and inter-day within 1.24% for rosamultin, within 3.40% for kaji-ichigoside F1, RSD%), recovery(within 97.57% for rosamultin, 99.16% for kaji-ichigoside F1), and stability(within 1.24% for rosamultin, 3.40% for kaji-ichigoside F1) were met the criteria of GPH 5-1. The results showed that the amount of rosamultin and kaji-ichigoside F1 was related to the commercial grades of the medicinal materials and nature environment of the producing area. Potentilla anserina L. originating from Qinghai-Tibet Plateau contains more rosamultin and kaji-ichigoside F1 than that from Yunnan-Guizhou Plateau. For the medicinal materials from the same origin, the lower commercial grade of medicinal materials, the higher amount of rosamultin and kaji-ichigoside F1 being contained.3. An SPE-HPLC-ESI-MS method was developed and validated in the present study. Linear calibration(r2=0.9990) curves were obtained over the concentration range of 5~500 ng/ml. Precision(intra- and inter-day within 7.67%, RSD%), accuracy(within 5.34%, RE %), matrix effect(LQC 111.9 ± 7.4%, MQC 88.9 ± 2.9%, HQC 88.9 ± 2.1%, and IS 101.4 ± 3.8%), recovery(LQC 91.0 ± 10.3%, MQC 87.7 ± 5.1%, HQC 84.0 ± 4.5%, and IS 85.4 ± 4.4%) and stability(within 13.41%, RE%) were met the criteria of the guideline on validation of bioanalytical methods by EMEA. The concentration-time profile results showed that rosamultin absorbed quickly after oral administration. The Tmax was only 0.2h, but the Cmax was only 332.81 ng/ml at an oral dosage of 60 mg/kg. The t1/2 was determined as 7.97 h. A double peak phenomenon was observed at about 4 h in different rats.Conclusion:1. The amount of rosamultin and kaji-ichigoside F1 was related to the grades of the medicinal materials and altitude of the producing area. Potentilla anserina L. originating from Qinghai-Tibet Plateau has more rosamultin and kaji-ichigoside F1. For the medicial materials from the same origin, the lower grade of medicinal materials, the higher amount of rosamultin and kaji-ichigoside F1.2. A simple, accurate and stable method was developed to dertermine rosamultin in the rat plasma. After oral administration, rosamultin absorbed quickly in rats but with low maximum plasma concentration, indicating poor cell membrane permeabilization of rosamultin. Double peak was observed in the drug – time profile, indicating that there might be an enterohepatic circulation in in vivo procedure. |
PDF Full Text Request