Anna Pitschmann1, Martin Zehl1,2, Elke Heiss1, Sodnomtseren Purevsuren3, Ernst Urban2, Verena M Dirsch1, Sabine Glasl1. 1. Department of Pharmacognosy, University of Vienna, Althanstraße 14, A-1090, Vienna, Austria. 2. Department of Pharmaceutical Chemistry, University of Vienna, Althanstraße 14, A-1090, Vienna, Austria. 3. School of Pharmacy and Biomedicine, Mongolian National University of Medical Science, Ulaanbaatar, PO 48 Box 111, Mongolia.
Abstract
INTRODUCTION: Leonurus sibiricus L. is regularly used in traditional Mongolian medicine including for the treatment of symptoms of diabetes mellitus. OBJECTIVES: To provide a validated quantitation method for the quality control of Leonurus sibiricus and to prove in vitro insulin-sensitisation, thereby supporting the traditional use of Leonurus sibiricus. METHODOLOGY: Pulverised Leonurus sibiricus material was either extracted with methanol or methanol:water (25:75, v/v). HPLC-CAD (charged aerosol detector) separations were performed on a Luna Phenyl-Hexyl column with water and acetonitrile (both modified with 0.1% formic acid) as mobile phase. Gradient elution was employed using theophylline as internal standard. Tentative peak identification was facilitated by HPLC-MS. Validation was carried out according to ICH (International Conference on Harmonisation) guidelines. Potential insulin-sensitisation of accordant extracts was assessed in glucose uptake experiments in C2C12 myocytes and protein tyrosine phosphatase 1B (PTP1B) enzyme assays. RESULTS: Thirty-six compounds were tentatively identified based on their retention times, UV spectra, MS fragments and data from literature. They comprise phenolcarboxylic acids, flavonoids, iridoid glycosides, and phenylpropanoids, among which acetylharpagide, ajugoside, lavandulifolioside, and verbascoside were selected for quantitation. The methanol extract contained 0.42% combined iridoids, and 1.58% combined phenylpropanoids. Validation showed good accuracy, intermediate precision and robustness. The methanol extract of Leonurus sibiricus led to a 1.5 fold increase in insulin-stimulated cellular glucose uptake and inhibition of PTP1B by 40% at a concentration of 10 µg/mL. CONCLUSION: HPLC-CAD analysis allowed sensitive quantitation of the selected marker compounds in Leonurus sibiricus, thereby providing a reliable tool for its quality control.
INTRODUCTION:Leonurus sibiricus L. is regularly used in traditional Mongolian medicine including for the treatment of symptoms of diabetes mellitus. OBJECTIVES: To provide a validated quantitation method for the quality control of Leonurus sibiricus and to prove in vitro insulin-sensitisation, thereby supporting the traditional use of Leonurus sibiricus. METHODOLOGY: Pulverised Leonurus sibiricus material was either extracted with methanol or methanol:water (25:75, v/v). HPLC-CAD (charged aerosol detector) separations were performed on a Luna Phenyl-Hexyl column with water and acetonitrile (both modified with 0.1% formic acid) as mobile phase. Gradient elution was employed using theophylline as internal standard. Tentative peak identification was facilitated by HPLC-MS. Validation was carried out according to ICH (International Conference on Harmonisation) guidelines. Potential insulin-sensitisation of accordant extracts was assessed in glucose uptake experiments in C2C12 myocytes and protein tyrosine phosphatase 1B (PTP1B) enzyme assays. RESULTS: Thirty-six compounds were tentatively identified based on their retention times, UV spectra, MS fragments and data from literature. They comprise phenolcarboxylic acids, flavonoids, iridoid glycosides, and phenylpropanoids, among which acetylharpagide, ajugoside, lavandulifolioside, and verbascoside were selected for quantitation. The methanol extract contained 0.42% combined iridoids, and 1.58% combined phenylpropanoids. Validation showed good accuracy, intermediate precision and robustness. The methanol extract of Leonurus sibiricus led to a 1.5 fold increase in insulin-stimulated cellular glucose uptake and inhibition of PTP1B by 40% at a concentration of 10 µg/mL. CONCLUSION: HPLC-CAD analysis allowed sensitive quantitation of the selected marker compounds in Leonurus sibiricus, thereby providing a reliable tool for its quality control.