Evaluation On Quality Of Tianma And The Pharmacokinetics Of Gastrodin After Oral Administration Of Tianma Extract In Rats

Tianma, the dried rhizome of Gastrodia elata B1., is an important and notable Chinese herbal medicine which has been used for over one thousand years in China. Modern pharmacology researches indicated that Tianma has the effects of anti-convulsion, conscious-sedation, anti-inflammatory, cardiovascular system and improving memory impairment. Tianma also has been used as a health supplement, ingredient included in cosmetics. In this thesis the evaluation on quality of Tianma and pharmacokinetics of bioactive compounds of Tianma were studied in order to provide scientific evidence for the quality control and rational administration of Tianma.1. Evaluation on quality of TianmaHPLC-DAD/MS techniques were used to study chemical fingerprint of Tianma. The major chemical components of Tianma were well separated under the established HPLC conditions and the method was validated and found to be suitable for the analysis of Tianma. Samples from 18 different habitats each habitat processed by 4 different methods were analyzed. The experiments were performed on a Zorbax Eclipse XDB C18 column (4.6mm×250mm, 5μm) with a gradient elution system which composed of acetonitrile and aqueous acetic acid. UV detection was used at 270 nm. The mass spectra was recorded using as ESI source in the negative mode with ion spray voltage at 3500V, source temperature at 335℃, gas spray at 8264 Pa and gas flow rate at 9 L/min. Data of chromatographic fingerprint were analyzed by the "Similarity Evaluation System for Chromatographic Fingerprint of TCM (Version 2004 A)" software to compare the quality of Tianma, which showed that samples from different habitats with the same processing method were of high similarity, though a few samples showed evident difference in fingerprint graphics. For Tianma samples with different processing methods, the contents of common peaks were different and the processing method of freezing to dry was better than others. By the above software, 10 common peaks were found in HPLC fingerprint as well. Among them 8 main constituents were characterized by their MS spectra and comparison with the reference standard and literatures. Meanwhile, HPLC-DAD/MS techniques were used to establish the metabolic fingerprint of Tianma. The rat plasma after oral administration of Tianma extract was analyzed and three main constituents in rat plasma were characterized by their MS spectra and comparison with the reference standard and literatures.In addition, a simple, reproducible and rapid HPLC method for quantification of Gastrodin (GAS) and p-hydroxybenzyl alcohol (HBA) in Tianma was developed. The conditions of HPLC analysis were same as those of chromatographic fingerprint analysis except the mobile phase, which consisted of acetonitrile and 0.1% aqueous acetic acid in isocratic elution mode with the ratio of 4.5 to 95.5(V/V). The method was validated and found to be suitable for the analysis of Tianma. 72 samples of Tianma from 18 differenthabitats with four different proceeding method were determined. The results of similarity analysis for chromatographic fingerprint were basically consistent with those of quantitative analysis.2. Pharmacokinetics of Gastrodin and p-hydroxybenzyl alcohol after oral administration of Tianma extract in rats by HPLC-MS methodIn this part, high performance liquid chromatography-electrospray ionization mass spectrometric (HPLC-MS) method was used to determine Gastrodin (GAS) and p-hydroxybenzyl alcohol (HBA) in rat plasma after oral administration of Tianma extract. Up to 200μL of plasma containing GAS, HBA and pyromucic acid (as internal standard, IS) were deproteinized with six volumes of methanol. Calibration curves showed linearity within the tested concentration range 2.00-200.00μg/mL for GAS and 0.832-104.00μg/mL for HBA in plasma with a correlation coefficient (r) greater than 0.9997. The limit of quantification of 2.00 and 0.83μg/mL for GAS and HBA had been achieved, respectively. The intra-day and inter-day precisions of the method were determined to be less than 17.82% for GAS and 10.21% for HBA. The recoveries were in the range of 78.3～86.88% with RSD less than 7.80% for GAS and 84.2～93.39% with RSD less than 4.30% for HBA, respectively. Evidence showed a rapid, simple and reproducible LC-MS assay was established to determine GAS and HBA in rat plasma.For Pharmacokinetics, the primary pharmacokinetic parameters of GAS after oral administration of Tianma extract were calculated by two-compartment model using WinNonlin 5.0.1 program. From the above data, we found that GAS in rat plasma was detected at 15-rain after oral administration, and reached to the C_max at 70 min. Another active constituent HBA could not be detected in rat plasma at the beginning time points by the established methods and detected only at 45, 70, 100 and 120rain after oral administration of Tianma extract. The results might be due to the very low content of HBA in Tianma extract. Additionally from the structures of GAS and HBA, HBA is the aglycone of GAS. HBA detected in rat plasma at later time points might be the metabolite of GAS.