Systematic Analysis of Post-Administrative Saiboku-To Urine by Liquid Chromatography to Determine Pharmacokinetics of Traditional Chinese Medicine

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Abstract
To disclose the mystery of a traditional Chinese medicine and to identify biologically active components, we analysed post-administrative urine for Saiboku-To, an anti-asthmatic Chinese herbal remedy. Systematic analysis of the components appearing in the urine was carried out by high-performance liquid chromatography (HPLC) with normal- and reversed-phase modes in combination. beta-D-glucuronidase-treated urine was subjected to rapid-flow fractionation (RFF) to achieve fractional extraction of lipophilic components with exhaustive recovery rates. The extracts were analysed by HPLC equipped with a multi-channel UV-detector. In the first stage of HPLC, we conducted a normal-phase mode run to find magnolol derived from Magnolia officinalis, as the most hydrophobic component showing minimum retention time among the urinary products of Saiboku-To. In the next stage, mobile phase solvent composition for reversed-phase HPLC was optimized so as to retain magnolol up to 60 min. Under these conditions, other Saiboku-To urinary products, which were more polar than magnolol, appeared within 60 min. Our HPLC method used marker compounds like magnolol and could indicate the terminal peak position on the reversed-phase chromatography. We found a total of eight components in the post-administrative Saiboku-To urine. Structure identification of the isolated pure materials was achieved using nuclear magnetic resonance (NMR)-, mass (MS)- and UV-spectra, and HPLC retention profiles. They were magnolol and 8,9-dihydroxydihydromagnolol stemming from M. officinalis, medicarpin and liquiritigenin from Glycyrrhiza glabra, baicalein, wogonin, and oroxylin A from Scutellaria baicalensis, and davidigenin of an unknown origin. The pharmacological mystery of Saiboku-To should be disclosed by resolving the pharmacokinetics and pharmacodynamics of these urinary products independently and synergistically.

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