Cardenolides content in wild Sardinian Digitalis purpurea L. populations.

In order to quantify the amounts of digitoxigenin and gitoxigenin in wild Sardinian Digitalis purpurea L. an easy extraction method and an high performance liquid chromatography (HPLC) analytical technique have been set up. The analyzed samples, stemming from six different locations, showed a great variability in glycoside content. The HPLC analyses carried out on 2-year-old plants of D. purpurea showed that the amounts of digitoxigenin and gitoxigenin ranged between 11.34 and 240.59 mg kg(-1) and 4.05 and 178.07 mg kg(-1), respectively, calculated on fresh material. Chemometric analyses, carried out considering different morphological characters, showed that correlations between morphological variations and glycoside content are poor.

Chromatography on lipophilic dextran gels for fractionation of low molecular weight compounds I: steroid digitonides.

A method for the separation of 3beta-hydroxysterols from other sterols is presented. The method involves precipitating 3beta-hydroxysterols as the digitonides. The digitonide is then decomposed and separated into components using chromatography on highly cross-linked lipophilic polysaccharide gels. The digitonin in the mother liquor is separated from other sterols using the same chromatographic procedure.

A note on the use of topical digitalis prior to William Withering.

Attention is called to the fact that, long before the systematization of oral digitalis therapy by Withering in the eighteenth century, the drug was applied to the skin by inunction, producing effects that can now be recognized as due to an overdosage of Digitalis glycosides. The history of digitalis is briefly reviewed: the drug appears not to have been known to Greek and Roman physicians, but by the Middle Ages was widely used in folk medicine. Contrary to current wisdom, there is a wealth of historical information suggesting that topically applied Digitalis glycosides are capable of exerting physiological activity. It is perhaps time to re-examine this feature, in view of the present-day general interest in transdermal medications.

Partition high-pressure liquid-chromatographic systems for the separation of digitalis glycosides of the cardenolide group.

Several multi-component liquid-systems have been investigated on silica gel SI-60 supports of particle size 10 mum. By using two solvent systems, it was possible to separate 14 digitalis glycosides, ranging from genins of relatively low polarity to the highly polar deacetyl-lanatosides. Solvents with good ultraviolet transparency at 220 nm (lambdamax. for the butenolide ring) were chosen in order to improve the sensitivity of detection. The technique should also permit the determination of by-products and degradation products of these drug substances. Detection limits are as low as 15 ng for a 5 mul injection, and separation times vary between 4 and 20 min. The reproducibility of the retention times and the baseline separations attainable make the systems suitable for quantitative work.

Isolation and quantitative determination of some cardioactive glycosides from Digitalis lanata by high-performance liquid chromatography.

A rapid extraction method followed by high-performance liquid chromatographic assay was developed for the quantitative determination of the cardioactive glycosides of Digitalis lanata. The leaf samples were extracted with water or aqueous alcohols. The simple extraction method gives a better yield than the methods described previously. Lanatoside C and its metabolites have been separated on a reversed-phase column with various mixtures of acetonitrile, methanol, and water as mobile phases for isocratic elution. Extraction and quantitative determination of lanatoside C and digoxin from a leaf sample require not more than 30 min.

A comparison of reversed-phase and partition high-performance liquid chromatography of some digitalis glycosides.

Comparison of the data for adsorption and reversed-phase chromatography of digitalis glycosides shows the complementary nature of the two modes of separation. The correct choice for a particular problem should make possible a rapid and good separation with simple isocratic systems. Detection limits vary between 10 and 100 ng per injection and permit the analysis of by-products even in low-dosage pharmaceutical formulations. Quantitation is easily possible with both chromatographic techniques using external standardization. The reproducibility for repetitive chromatograms is about 1% relative standard deviation for manual injections by loop injectors and is even significantly better for automatic injection. Reversed-phase chromatography can offer some advantages with regard to sample preparation of pharmaceutical formulations.

Induction of digoxin-like material production, and the digoxin binding in the unicellular organism Tetrahymena by digitoxin.

Thin layer chromatographic, and laser-confocal microscopic analyses with a monoclonal antibody to digoxin also displaying high affinity to digoxigenin, were used to determine the presence and localization of cardioactive glycosides. Tetrahymena pyriformis was found to possess digitoxigenin-like material, but digoxin, digitoxin, digoxigenin, gitoxin and lanatoside C were not detected. Digitoxin treatment elicited the appearance of a digoxin-like material in the progeny generations. Digoxin was taken up by untreated Tetrahymena, especially strongly 24 h after digitoxin treatment. While the cardenolide was localized in vesicles of the cell body in untreated Tetrahymena, the engulfed digoxin appeared in the epiplasmic layer and also in the cilia after digitoxin pretreatment. Digoxin pretreatment did not increase digoxin uptake. These data indicate that Tetrahymena has: (1) the capacity to discriminate between closely related molecules; (2) the ability to induce digoxin-like material production; and/or (3) enzymes that can effect a digitoxin-digoxin transformation.

A molecular recognition hypothesis for nonpeptides: Na+ K+ ATPase and endogenous digitalis-like peptides.

The molecular recognition hypothesis for peptides is that binding sites of ligands and their receptors are encoded by short, complementary segments of DNA. A corollary hypothesis for nonpeptide ligands posited here is that peptide replicas may be encoded by the DNA segment complementary to the receptor binding sites for nonpeptides. This corollary was tested for digitalis. a family of cardiotonic and natriuretic steroids including ouabain. A hexapeptide (ouabain-like peptide, OLP) complementary to a ouabain binding site on sodium potassium dependent adenosine triphosphatase (Na+ K+ ATPase) exhibited activity in a digitalis bioassay. Antisera to the complementary peptide (OLP) stained the neurohypophysis in an immunocytochemical procedure. The complementary peptide was found to share an identical 4-amino acid region with the 39-amino acid glycopeptide moiety of the vasopressin-neurophysin precursor. This glycopeptide was isolated from pituitary extracts; it exhibited digitalis-like activity in the submicromolar range and cross-reacted with complementary peptide antibodies. Another digitalis-like substance with high activity also was detected in the extracts. These results demonstrate that the vasopressin-neurophysin glycopeptide has digitalis-like activity. Moreover, the findings are consistent with the hypothesis that peptide mimetics of nonpeptides are encoded in the genome.

Purification and characterization of endogenous digitalis-like substance (DLS) from pig heart.

This study was undertaken to further purify and characterize a crude DLS-containing fraction isolated from left ventricular tissue of pig heart. The crude fraction, on purification, separated into three fractions designated as C1, C2 and C3. Only fraction C1 produced positive inotropic response on isolated perfused guinea pig heart. Fraction C1 specifically inhibited rat heart Na+, K(+)-ATPase, whereas fractions C2 and C3 were found to be non-specific inhibitors. Studies with cardiac Na+, K(+)-ATPase, its interaction in the presence of increasing concentration of [K+] and competitive displacement of [3H]-ouabain binding, demonstrated fraction C1 to have ouabain-like properties. C1 also inhibited dog kidney Na+, K(+)-ATPase activity in a dose-dependent manner and was found to be devoid of catecholamines, peptides and lipids. Fraction C1 was found to be heat sensitive and its inotropic activity was destroyed on ashing at 500 degrees C. These studies provide further evidence for the presence of an endogenous DLS in mammalian heart.