| The present study was aimed to investigate the Tianshu Capsule which was used to treat or prevent migraine. In this paper, the chemical basis, quality control method, pharmacokinetics and metabonomics of Tianshu Capsule were studied systematically, so as to provide enough information for its clinical usage. The contents are as follows;1. Extraction separation and structural identification of the indexing chemical constituents in Tianshu Capsule The chemical constituents of Chuanxiong Rhizoma and Gastrodia Elata Rhizome were systematically extracted, separated and purified by various chromatography technologies, and finally 10 compounds were achieved and identified by different chemical methods, current spectrum technology and references, which were fully characterized as ferulic acid, 6,7-di-hydroxyligustilide, 4-hydroxy-3-butylphthalide,senkyunolide C, caffeic acid, gastrodin,p-hydroxybenzyl alcohol,4-hydroxybenzaldehyde,p-hydroxybenzoic acid and 4,4'-dihydroxybenzyl sulfone. These compounds were suitable as standard substances for quantitative and pharmacokinetic purposes,2. Quality control study of Tianshu Capsule A high performance liquid chromatographic(HPLC) method with diode array detection (DAD ) was developed and validated to simultaneously determine 6 compounds including gastrodin, p-hydroxybenzyl alcohol,4,4'-dihydroxybenzyl sulfone, ferulic acid, 6,7-di-hydroxyligustilide and 4-hydroxy-3-butylphthalide in co-boiling extracts and pooled individual extracts of Chuanxiong Rhizoma and Gastrodia Elata Rhizome in different ratios. The interaction of herbs was observed when the herbs were co-boiled and pooled after boiled individually in different ratio(chuanxiong rhizoma: gastrodia elata Rhizome =4:1 and 1:1, g/g). The results indicated that the content of gastrodin, p-hydroxybenzyl alcohol and 4,4'-dihydroxybenzyl sulfone from rhizoma ligustici wallichii and 4-hydroxy-3-butylphthalide from rhizoma gastrodiae trended to increase,with a significant increase in the co-boiled decoction, suggesting co-boild decoction could increase the solubility of index comments in Tianshu Capsule based on the chemical base to calrify the meaning of such method. A RP-HPLC method under a changed UV wavelengths and gradient elution mode was developed and validated to simultaneously determine gastrodin,ferulic acid and 6,7-dihydroxyligustilide in Tianshu capsule. The method is accurate, sensitive and reliable for determination of gastrodin, ferulic acid and 6,7-dihydroxyligustilide. Compared with the Chinese Pharmacopoeia (2010), the method added a new marker ingredient into the assay content and improved the quality standard of Tianshu Capsule.3. The pharmacokinetic study of index conments for Tianshu Capsule in rat plasma A LC-MS method was developed and validated for simultaneous determination of 6,7-di-hydroxyligustilide and gastrodin in rat plasma, and which was subsequently applied in the pharmacokinetic analysis of co-boiling extracts and single extracts of Chuanxiong Rhizoma and Gastrodia Elata Rhizome in different ratio. Puerarin was selected as internal standard. The plasma was pretreated by acetonitrile:methanol (2:1, v/v). The analytes were separated on a Kromasil C18 column with a gradient elution program and detected without interference in the selected ion monitoring mode with positive electrospray ionization. The linear range was 0.010-10.0?g/mL for 6,7-di-hydroxyligustilide and 0.025-25.0 ?g/mL for gastrodin with the limit of quantitation of 0.01 and 0.025?g/mL, respectively. The extraction recoveries were more than 80% for the all analytes. The method including selectivity, matrix effect, precision and accuracy, stability were all within the scope of biological sample analysis. A rapid, sensitive and specific liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method was used for quantification of ferulic acid in rat plasma after oral administration of above extracts. The pharmacokinetic parameters indicated that gastrodin, 6,7-di-hydroxyligustilide and ferulic acid are absorbed rapidly and reached a maximum concentration soon which was consistent with the clinical requirements for the rapid relieving the symptoms of migraine. The results also indicated that there were obvious differences in the pharmacokinetic behaviors after oral administration of the co-boiling extracts and single extracts.4. The metabonomic study of Tianshu Capsule in migraine model rats. The migraine rat model was produced by glyceryltrinitrate infusion, via the validation of scratching head times and calcitonin-gene-related peptide (CGRP) after 2 h. A UPLC-MS method was developed and applied to investigate the metabonomics. An ACQUITY UPLC TM BEH C18 column was used with a gradient mobile phase of acetonitrile: 0.1% formic acid water, Electrospray ionization(ESI) source was operated in the positive ion mode by multiple reaction monitoring (MRM)mode within the scan scope of m/z 150?1200. The validated method was used to analyze the brain, serum and urine from control group, model group, Tianshu Capsule group and flunarizine group. The analytical data were processed via principle component analysis (PCA). PCA score piots of analytical data indicated the general differences among different groups and the influence of Tianshu Capsule on migraine. The dominant metabolites obtained from loading plot of Masslynx were the differential metabolites among different groups, ten of which were conformed. They are 16:0 LPC, 18:0 LPC, 18:1 LPC, 14:0 PC, 16:0 PC and cholic acid from brain and16:0 LPC, 18:0 LPC, 18:1 LPC, 18:2 LPC, 20:4 LPC and cholic acid from serum,xanthurenic acid, kynurenic acid, hippuric acid, phenylacetylglycine and hexadecas-phinganine from urine. The mechanism of pathological changes may be elucidated with the up- or down-regulated metabolic pathways. |
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