The Tissue Distribution of Four Major Coumarins after Oral Administration of Angelicae Pubescentis Radix Extract to Rats Using Ultra-High-Performance Liquid Chromatography

Abstract

Angelicae pubescentis radix (APR) is widely applied in treating rheumatoid arthritis in China. Coumarins are the major active compounds of APR extract including columbianetin, columbianetin acetate, osthole, and columbianadin. The in vivo behavior of the four major coumarins of APR has not been systematically reported. A feasible and reliable ultra-performance liquid chromatography (UPLC) method was established and validated for the quantification of the above four coumarins in rat various tissues (including heart, liver, spleen, lung, kidney, uterus, ovary, and muscle) after oral administration of APR extract. The separation was implemented on a Waters ACQUITY BEH C18 column (4.6 mm × 100 mm, 1.7 μm) with gradient mobile phase comprising acetonitrile-water (with 1mM formic acid) at a flow rate of 0.3 mL/min. The tissue homogenate samples were prepared by liquid-liquid extraction with ethyl acetate. The calibration curves were linear in the range of 1.6-20000 ng/mL for four coumarins with the lower limit of quantitation of 1.6 ng/mL in rat tissues. The intraday and interday precisions and recoveries were all within 80-100% with the relative standard deviations (RSDs) which were all less than 10.9%. The method was successfully applied to the tissue distribution research after oral administration of 6.0 g/kg APR extract to rat. The results revealed that the tissues distributions of four coumarins were in the liver, followed by the ovary, uterus, kidney, lung, heart, spleen, and muscle.

Figure 1

The UHPLC-PDA chromatograms with APR extract sample (a) and standard solution of four compounds (b). 1: columbianetin, 2: columbianetin acetate, 3: osthole, 4: columbianadin.

Figure 2

Chemical structures of columbianetin, columbianetin acetate, osthole, and columbianadin.

Figure 3

Representative tissue chromatogram of (a) blank heart tissue, (b) blank heart tissue spiked with the standard compounds at LLOQ, and (c) real samples after administration of APR extract. 1: columbianetin, 2: columbianetin acetate, 3: osthole, 4: columbianadin.

Figure 4

The tendency graph of columbianetin, columbianetin acetate, osthole, and columbianadin in eight tissues at 4, 6, and 8 h after oral administration of APR extract (n=6).