Profiling and structural analysis of cardenolides in two species of Digitalis using liquid chromatography coupled with high-resolution mass spectrometry.

Plants of the Digitalis genus contain a cocktail of cardenolides commonly prescribed to treat heart failure. Cardenolides in Digitalis extracts have been conventionally quantified by high-performance liquid chromatography yet the lack of structural information compounded with possible co-eluents renders this method insufficient for analyzing cardenolides in plants. The goal of this work is to structurally characterize cardiac glycosides in fresh-leaf extracts using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) that provides measured accurate mass. Fragmentation of cardenolides is featured by sequential loss of sugar units while the steroid aglycone moieties undergo stepwise elimination of hydroxyl groups, which distinguishes different aglycones. Using a reverse-phase LC column, the sequence of elution follows: diginatigenin→digoxigenin→gitoxigenin→gitaloxigenin→digitoxigenin for cardenolides with the same sugar units but different aglycones. A linear range of 0.8-500 ng ml-1 has been achieved for digoxigenin, ß-acetyldigoxin, and digitoxigenin with limits of detection ranging from 0.09 to 0.45 ngml-1. A total of seventeen cardenolides have been detected with lanatoside A, C, and E as major cardenolides in Digitalis lanata while seven have been found in Digitalis purpurea including purpurea glycoside A, B, and E. Surprisingly, glucodigifucoside in D. lanata and verodoxin and digitoxigenin fucoside in D. purpurea have also been found as major cardenolides. As the first MS/MS-based method developed for analyzing cardenolides in plant extracts, this method serves as a foundation for complete identification and accurate quantification of cardiac glycosides, a necessary step towards understanding the biosynthesis of cardenolide in plants.

Quantitative determination of cardiac glycosides in Digitalis lanata leaves by reversed-phase thin-layer chromatography.

An analytical method for the determination of cardiac glycosides in Digitalis lanata leaves by reversed-phase thin-layer chromatography (RP-TLC) was developed. The procedure consisted of extraction of dry leaf powder with 50% methanol and clean-up by Sep-Pak cartridges prior to RP-TLC analysis. RP-TLC was performed on an octadecylsilyl bonded silica gel plate, using a developing solvent of acetonitrile-methanol-0.5 M NaCl (1:1:1) for primary glycosides and acetonitrile-methanol-0.5 M NaCl (12:7:9) for secondary glycosides. The plate was scanned with a reflectance densitometer at 225 nm. The quantitation was carried out by the internal standard method. The present method is reliable and relatively simple for the determination of cardiac glycosides in Digitalis lanata leaves.

Quantitative HPLC analysis of cardiac glycosides in Digitalis purpurea leaves.

An analytical method for the determination of cardiac glycosides in Digitalis purpurea leaves by hplc was developed. Quantitation was carried out by the incorporation of lanatoside A as an internal standard. The present method is sufficiently precise and relatively simple.

Cardiac glycosides in partly submerged shoots of Digitalis lanata.

Shoot cultures were established from axillary buds (11 strains) or seeds (1 strain) of individual Digitalis lanata Ehrh. plants and propagated partially submerged in liquid medium. Five of these shoot culture strains were characterized with regard to their growth and cardenolide content. The cultures were observed for more than one year and found to be relatively stable with regard to their growth and cardenolide spectrum and yield. The strains examined differed in terms of their total cardenolide yield, which ranged from about 30 nmol g DW-1 to almost 1000 nmol g DW-1. Cardenolide content was correlated with leaf size and development. Depending on the strain investigated up to ten different cardenolides could be detected by HPLC. The main cardenolides were identified by comparing HPLC and TLC results with those of authentic samples and chemical degradation as being the mono- and diglycosides glucodigifucoside, glucoverodoxin, odorobioside G, and odoroside H; minor amounts of digitalinum verum and glucoevatromonoside were also found. In addition, the tetrasaccharides lanatoside A and C were present. The shoots were cardenolide-free when cultivated in the dark for more than 30 weeks, but regained their characteristic cardenolide profile when transferred back to light. For the dark cultivation of chlorophyll-free cultures a medium containing 3.5% glucose was found to be optimal.

High-performance liquid chromatographic determination of secondary cardiac glycosides in Digitalis purpurea leaves.

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.

Structural basis of steroid compounds interaction with "digitalis"-receptor sites of Na,K-dependent ATPase.

Twenty-two Steroid molecules have been tested for the inhibition Na,K-dependent ATPase at 10(-7)-10(-4) M concentrations. At the 10(-5) M concentration of the investigated molecules, inhibition ranged from 8 to 36%. To explain the structure-inhibition % relationship, we determined the value of heteropolarity or biphilicity moment of these molecules. This value would appear to be dependent on the space location and hydrophilicity of the molecule elementary fragments, and to the degree of their water accessibility; however, it is independent of the hydrophilicity of the molecules as a whole. On the basis of the obtained data, details of Na,K-ATPase digitalis-receptor structure and the mechanism of the glycoside-receptor interaction are discussed.

[Characterization of an endogenous factor with digitalic activity in the guinea pig heart (II): Binding to a receptor]. / Caracterización de un factor endógeno con actividad digitálica en el corazón de cobayo (II): Unión al receptor.

In this work we continue the studies on the presence of an endogenous digitalis-like factor in mammals. A water extract from guinea pig heart was partially purified by liquid chromatography. The extract displaced (3H)ouabain specific binding to guinea pig heart Na,K-ATPase yielding a displacement curve parallel to that observed when digoxin was used as displacing agent. The data suggest that the activity of the extract was due to the presence of a factor that recognizes the digitalis binding site but not to ions nor lipids. Furthermore, the extract cross-reacted with antidigoxin antibodies in a manner similar to the specific antigens. These results support the hypothesis of the existence of an endogenous ligand of the digitalis receptor in mammals that could be a physiological regulator of the sodium pump.

[Production of an anti-digitalin monoclonal antibody (author's transl)]. / Production d'un anticorps monoclonal anti-digitaline.

Anti-digitalin monoclonal antibodies were obtained by hybridization of lymphoid cells from mice immunized with the drug and the 8653 myeloma cells from BALB/c Mice. Two of these antibodies were submitted to immunochemical analysis which revealed that they belonged to IgM and IgGl classes, and the high affinity of one of them (1,2 x 10(9) M(-1)). The availability os such antibodies opens new perspectives for the treatment of digitalin intoxication and the possibility of establishing a standardized radioimmunoassay.

Digitalis-like biological activity and immunoreactivity in chromatographic fractions of rabbit adrenal extract.

Dichloromethane extract of rabbit adrenals was subjected to thin-layer chromatography (Kieselgel 60F-254, MERCK) and digoxin immunoreactivity and digitalis glycoside-like biological activity were determined in the fractions by homogeneous enzymoimmunoassay and by inhibition of 86Rb uptake by red blood cells in vitro, respectively. Both the immunoreactivity and the biological activity maximum were present in two fractions which moved (in chromatography) between the corticosterone and deoxycorticosterone and showed a different RF from the deoxycorticosterone and aldosterone. In association with previous findings on digoxin-like immunoreactivity in the serum of animals with a cardiac overload, the possible existence of a cardiotropic steroid of adrenocortical origin is suggested.

Reverse phase thin layer chromatographic procedure for identification of digoxin and related fluorescing substances.

A one-step method was developed to replace the current USP monograph procedrues for identification of digoxin and determination of related fluorescing substances. The new method is much quicker than the previous tests, which required paper chromatography and fluorometric measurements, respectively. Using reverse phase silica gel plates (C18 bonded to silica gel), digoxin was separated from digitoxin, gitoxin, and digoxigenin mono-digitoxoside, and partially resolved from digoxigenin bis-digitoxoside. A digoxin reference standard is used to correlate Rf values for identification, and a dilute solution of gitoxin is also co-spotted to give a maximum intensity limit for related fluorescing substances.

Digoxin degradation in acidic dissolution medium.

The release of digoxin and its simultaneous conversion to digoxigenin bisdigitoxoside, digoxigenin monodigitoxoside, and digoxigenin in a USP dissolution test medium were followed by high-pressure liquid chromatography. Two products, Tablets A and B, were manufactured by solvent deposition and simple blending methods, respectively. Tablet A released digoxin faster than Tablet B in distilled water and in artificial intestinal juice, and no decomposition was observed. In the USP dissolution test medium, the rate of hydrolysis to digoxigenin bisdigitoxoside was almost equal to that of hydrolysis to digoxigenin monodigitoxoside, and a comparatively large formation rate of digoxigenin was observed. Concentrations of digoxin and its decomposition products were described by differential equations that included dissolution rates of digoxin (rapidly dissolving digoxin and digoxin crystals) and an apparent hydrolysis rate. In the earlier stage of dissolution, hydrolysis was rate determining; in the later stage, dissolution became the rate-determining step for overall digoxin degradation. To suppress digoxin hydrolysis in the USP dissolution test medium, a developmental formulation study was performed. The incorporation of magnesium oxide and magnesium hydroxide-aluminum hydroxide in the tablet formulations inhibited digoxin hydrolysis by 15.3 and 14.5%, respectively, after dissolution for 30 min without serious delay of drug release.