A rapid quantitative ¹H NMR analysis of kinsenoside and other bioactive principles from: Anoectochilus formosanus

Anoectochilus formosanus is a famous traditional Taiwanese folk medicine. Kinsenoside (1), one of the major active components in A. formosanus, exhibits hepatoprotective, antihyperliposis and antiflammatory activities. In this study, a proton nuclear magnetic resonance (¹H NMR) method was developed for rapid quantitative analysis of several bioactive major constituents, including kinsenoside (1), 3-(R)-3-β-d-gluco-pyranosyloxy-4-hydroxybutanoic acid (2), 1-O-isopropyl-β-d-gluco-pyranoside (3), 4-(β-d-gluco-pyranosyloxy)benzyl alcohol (4) and benzoic acid (5) in plant samples and commercial products of A. formosanus. Interferences were removed by a simple Diaion HP-20 column chromatography. In the ¹H NMR spectrum, the signals of compounds 1 (δ 2.88), 2 (δ 2.47), 3 (δ 1.12), 4 (δ 7.05) and 5 (δ 7.84) were selected and integrated as the target peaks, separated well from other signals in D₂O. The developed ¹H NMR method provides a rapid and simple quantitative method without any derivatization, pH control or addition of NMR shift reagent, and the analytical solvent system is very safe, convenient and simple.