| Dioscorea nipponica Mak. is a native perennial herb distributed throughout China. Its rhizomes have been known in traditional Chinese medicine (TCM ) as Chuanlongshuyu,which used for the treatment of gastropathy,anthrax,rheumatic heart disease and rheumarthritis. Chuanlongshuyu is one of the important folk medicines in TCM.In order to isolate the active constituents in Chuanlongshuyu, various chromatographic techniques were performed. Six compounds were isolated and identified with IR, NMR and MS data and physical-chemical properties. They were diogenhuβ-itosterol,dioscin,benzoic acid,protodioscin,trillin. The investigation on reducing blood liquids and free radical effects of these compounds were investigated (in vivo). It was suggested that four steroid sa ponins possessed reducing activity to blood liquids and free radical.Dioscin has been reporting as one of the effective components. We established a method for the determination of dioscin in Chuanlongshuyu and Dioscorea by RP-HPLC. The separation was achieved on a Xterra? Cis column(250 mm×4.6 mm, 5μm)using acetonitrile-water (55:45) as the mobile phase at a flow rate of 1.0 mL·min-1, 203 nm as the detection wavelength. Dioscin was well-separated. The linear range of dioscin was 0.1～0.8 mg·mL-1 (r = 0.9999). The average recovery was 97.9% with RSD of 2.0% (n = 9) .The assay was found to be simple and accurate to measure the concentrations of dioscin in Chuanlongshuyu and its preparation.To establish a method for the determination of protodioscin in Chuanlongshuyu by RP-HPLC . The chromatographic condition included a Xterra? C18 column (250 mm×4.6 mm i.d, 5μm) and the mobile phase consisting of acetonitrile-water (27:73, v/v) and the absorbance was monitored at 203 nm. The calibration curve was linear over the range of 0.02～0.6 mg·mL-1 for protodioscin (r=0.9999, n=6). The average recovery was 98.0% (RSD=1.5%). The assay was found to be simple and accurate to measure the concentrations of protodioscin in Chuanlongshuyu and its preparation.We have established a rapid, sensitive and selective LC-ESI-MS/MS method to measure protodioscin in rat plasma and investigated the pharmacokinetics of protodioscin after intravenous administrations. Plasma samples (40μL) were prepared after plasma protein precipitation, and a 20μL aliquot of the supernatant was injected directly onto a analytical column (50 mm×2.0 mm i.d.) with an mobile phase consisted of acetonitrile-water-formic acid (80:20:0.1, v/v/v). Analytes were detected with a LC-ESI-MS/MS system in positive multiple reaction monitoring mode (m/z 1032.6 (precursor ion) to m/z 869.7 (product ion) for protodioscin; m/z 577.1 (precursor ion) to m/z 253.1 (product ion) for trillin(internal standard). The sensitivity of the method was 20 ng/mL and a linear range of 20-125000 ng/mL. The intra-and inter-day relative standard deviation (RSD) across three validation runs over the entire concentration range was＜8.0%. Accuracy determined at three concentrations (50, 5000 and 50000 ng/mL for protodioscin) ranged from 0.2 to 1.8% as terms of relative error (R.E.). Each plasma sample was chromatographed within 3.5 min. This LC-ESI-MS/MS method allows accurate, high-throughput analysis of protodioscin in small amounts of plasma and possibly other biological samples. |
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