Quantitative fluorimetric determination of the main cardiac glycosides in Digitalis purpurea leaves.
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The capability of digitalis glycoside-specific antisera to reverse the glycoside-induced inhibition of (Na+ + K+)-ATPase activity was examined. The antisera-induced reversal of the ATPase inhibition caused by digoxin was considerably slower (k-1 = 0.1177 . 10-3 .S-1) than the reversal caused by dihydrodigoxin and dihydrodigitoxin (k-1 = 4.462 . 10-3 . S-1 and K-1 = 3.609 .10-3 . S-1, respectively). In addition, the dissociation rate constants of the ouabain-induced dissociation of the various glycosides from the glycoside-ATPase complex were determined: The dissociation rate constants of the antisera-induced restoration of enzyme activity and that of the ouabain-induced dissociation of glycosides from the glycoside-enzyme complex were not significantly different.
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An analytical method for the determination of secondary cardiac glycosides in Digitalis purpurea leaves by high-performance liquid chromatography (HPLC) is described. The procedure consisted of extraction of dry leaf powder with ethanol-chloroform (2:1) and clean-up by Sep-Pak cartridges prior to HPLC analysis. HPLC was performed on an octylsilyl bonded silica column, using acetonitrile-methanol-water (4:4:5) for trisdigitoxosides and acetonitrile-methanol-water (8:30:43) for strospeside; the effluent was monitored by ultraviolet detection (at 220 nm). Quantitation of these cardiac glycosides was carried out by the internal standard method. The amounts of digitoxin, gitoxin, gitaloxin and strospeside per 100 mg of dry leaf powder were estimated to be 22.6, 14.0, 54.7 and 1.9 micrograms, respectively. The method is sufficiently sensitive and reproducible to assay secondary glycosides in Digitalis purpurea leaves.
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