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[Estimation of the effects of cardenolides. 1. Relation between molecular physical parameters and parameters of action of cardenolides].

Knowing the modell developed by Repke and co-workers for the cardenolide-receptor-interaction the aim of this study was to find quantitative relations between molecular physical properties of cardenolides and their molecular-biologically defined effect, which are fundamentally useful to estimate the effect of still nonsynthesized cardenolide derivatives. The statistically significant relations (alpha = 0.05) between molecular physical parameters, which are derived from the dipole moment vector, and the molecular-biologically defined effect was found by means of regression analysis.

Adenosine Triphosphatases↗

Cardenolide analogues. 3. A fast thin layer chromatographic separation of cardenolide diastereomers.

Cardenolide diasteromeric mixtures may be analytically separated on 2.5 x 6.5 cm x 0.25 mm pre-coated silical gel 60 F-250 (EM) tlc plates (cut from 20 x 20 cm plates). Three successive developments in mixtures of CH2Cl2-EtOAc-MeOH achieve complete separation of diastereomers in less than 15 minutes, and 2--6 micrograms of sample is sufficient. This procedure facilitated the separation of several diastereomeric mixtures, including 20(R) and 20(S)-20,22-dihydrocardenolides. A single development produces separations sufficient for separating starting materials and products of common cardenolide types of reactions.

Cardenolides↗

Synthesis of cardenolide glycosides and putative biosynthetic precursors of cardenolide glycosides.

A rapid and efficient procedure for glycosylation of steroids was established using a modified Koenigs-Knorr procedure. Peracetylated beta-glycosides were synthesized by reaction of cardenolides, various pregnanes and 23-nor-5,20(22)E-choldienic acid at room temperature with the peracetylated 1-bromo derivatives of D-glucose, D-galactose, D-fucose and cellobiose. Subsequent deprotection was performed by alkaline hydrolysis with sodium methoxide. Structures of the respective glycosides were established by NMR techniques. The complete protocol was shown to be non-destructive at all stages to the sugar moiety and the steroidal nucleus. The gamma-unsaturated lactone ring of the cardenolides was shown to remain intact and no formation of C-14 unsaturated compounds was observed.

Cardenolides↗

Antidotes for acute cardenolide (cardiac glycoside) poisoning.

BACKGROUND: Cardenolides are naturally occurring plant toxins which act primarily on the heart. While poisoning with the digitalis cardenolides (digoxin and digitoxin) are reported worldwide, cardiotoxicity from other cardenolides such as the yellow oleander are also a major problem, with tens of thousands of cases of poisoning each year in South Asia. Because cardenolides from these plants are structurally similar, acute poisonings are managed using similar treatments. The benefit of these treatments is of interest, particularly in the context of cost since most poisonings occur in developing countries where resources are very limited. OBJECTIVES: To determine the efficacy of antidotes for the treatment of acute cardenolide poisoning, in particular atropine, isoprenaline (isoproterenol), multiple-dose activated charcoal (MDAC), fructose-1,6-diphosphate, sodium bicarbonate, magnesium, phenytoin and anti-digoxin Fab antitoxin. SEARCH STRATEGY: We searched MEDLINE, EMBASE, the Controlled Trials Register of the Cochrane Collaboration, Current Awareness in Clinical Toxicology, Info Trac, www.google.com.au, and Science Citation Index of studies identified by the previous searches. We manually searched the bibliographies of identified articles and personally contacted experts in the field. SELECTION CRITERIA: Randomised controlled trials where antidotes were administered to patients with acute symptomatic cardenolide poisoning were identified. DATA COLLECTION AND ANALYSIS: We independently extracted data on study design, including the method of randomisation, participant characteristics, type of intervention and outcomes from each study. We independently assessed methodological quality of the included studies. A pooled analysis was not appropriate. MAIN RESULTS: Two randomised controlled trials were identified, both were conducted in patients with yellow oleander poisoning. One trial investigated the effect of MDAC on mortality, the relative risk (RR) was 0.31 (95% confidence interval (CI) 0.12 to 0.83) indicating a beneficial effect. The second study found a beneficial effect of anti-digoxin Fab antitoxin on the presence of cardiac dysrhythmias at two hours post-administration; the RR was 0.60 (95% CI 0.44 to 0.81). Other benefits were also noted in both studies and serious adverse effects were minimal. Studies assessing the effect of antidotes on other cardenolides were not identified. One ongoing study investigating the activated charcoal for acute yellow oleander self-poisoning was also identified. AUTHORS' CONCLUSIONS: There is some evidence to suggest that MDAC and anti-digoxin Fab antitoxin may be effective treatments for yellow oleander poisoning. However, the efficacy and indications of these interventions for the treatment of acute digitalis poisoning is uncertain due to the lack of good quality controlled clinical trials. Given pharmacokinetic differences between individual cardenolides, the effect of antidotes administered to patients with yellow oleander poisoning cannot be readily translated to those of other cardenolides. Unfortunately cost limits the use of antidotes such as anti-digoxin Fab antitoxin in developing countries where cardenolide poisonings are frequent. More research is required using relatively cheap antidotes which may also be effective.

Acute Disease↗

Density-dependent reduction and induction of milkweed cardenolides by a sucking insect herbivore.

The effect of aphid population size on host-plant chemical defense expression and the effect of plant defense on aphid population dynamics were investigated in a milkweed-specialist herbivore system. Density effects of the aposematic oleander aphid, Aphis nerii, on cardenolide expression were measured in two milkweed species, Asclepias curassavica and A. incarnata. These plants vary in constitutive chemical investment with high mean cardenolide concentration in A. curassavica and low to zero in A. incarnata. The second objective was to determine whether cardenolide expression in these two host plants impacts mean A. nerii colony biomass (mg) and density. Cardenolide concentration (microgram/g) of A. curassavica in both aphid-treated leaves and opposite, herbivore-free leaves decreased initially in comparison with aphid-free controls, and then increased significantly with A. nerii density. Thus, A. curassavica responds to aphid herbivory initially with density-dependent phytochemical reduction, followed by induction of cardenolides to concentrations above aphid-free controls. In addition, mean cardenolide concentration of aphid-treated leaves was significantly higher than that of opposite, herbivore-free leaves. Therefore, A. curassavica induction is strongest in herbivore-damage tissue. Conversely, A. incarnata exhibited no such chemical response to aphid herbivory. Furthermore, neither host plant responded chemically to herbivore feeding duration time (days) or to the interaction between herbivore initial density and feeding duration time. There were also no significant differences in mean colony biomass or population density of A. nerii reared on high cardenolide (A. curassavica) and low cardenolide (A. incarnata) hosts.

Animals↗

Relationship between the inotropy speeds in guinea-pig myocardium and lipophilic character of cardenolides and ericaceous toxins.

Interrelation between lipophilic characters and speeds of positive inotropic effect (PIE) of cardenolides and ericaceous toxins was studied by determining both parameters of lipophilicity and inotropy speeds. The lipophilic characters of 8 kinds of cardenolides, evaluated from Rm or log k' values by means of thin layer chromatography (Rf) or high performance liquid chromatography (retention time), increased in the order of ouabain, digoxin, digitoxigenin, digitoxigenin-monodigitoxoside, digitoxigenin-bis-digitoxoside, digitoxin, alpha-acetyl-digitoxin, triacetyl-digitoxin. Lipophilic character, evaluated from Rm values of 6 kinds of ericaceous toxins, was in decreasing order of 10S-grayanotoxin II, 6-acetyl grayanotoxin I, asebotoxin I, grayanotoxin I, asebotoxin III, asebotoxin X. The speed with which PIE developed was evaluated from the time to half maximum PIE (T50) of cardenolides and ericaceous toxins at a pD2 concentration. The speed of positive inotropy of cardenolides was independent of their concentration tested in the range from half to twice the concentration of pD2, while the speeds of PIE of ericaceous toxins depended on their concentration in the same range used in case of cardenolides. Inotropy speed of these two classes of cardiotoxins correlated well with the lipophilic character: a) In the case of cardenolides, a positive and close correlation (r = 0.98) was observed between T50 and Rm. The more lipophilic the cardenolides, the more time was required to reach the fully development of PIE. b) In contrast, a negative correlation (r = -0.82, between Rm and T50) was obtained in the case of ericaceous toxins; Toxins with more lipophilic nature caused faster development of PIE. The present results can be interpreted to mean that the PIE receptor for cardenolides in myocardial cells is located on the outer surface of the sarcolemma, while that for ericaceous toxins is located on the inside of the myocardial cell.

Animals↗

Seasonal cardenolide production and Dop5betar gene expression in natural populations of Digitalis obscura.

Productivity variations and seasonal fluctuations of cardenolides have been studied in 10 natural populations of Digitalis obscura distributed in three bioclimatic belts. Main cardenolides in D. obscura plants are those of the series A and such predominance (ca. 80-85%) over the series B metabolites is independent of the population studied or the degree of maturity of the leaves. Primary glycosides represent ca. 50-60% of total cardenolides; this percentage did not vary among populations or with the leaf age but increased in summer and decreased in winter. A correlation analysis between plant biomass and cardenolide content showed a positive relationship of these parameters, which, according to the bioclimatic distribution of the populations, suggests that certain environmental conditions may cause marked decreases in plant biomass together with a reduction in productivity. Cardenolide contents changed in the timecourse of the four seasons as a multiple response to distinct plant and/or environmental factors. The lowest production was recorded in May, followed by a fast cardenolide accumulation in summer, a decreasing phase in autumn, and a stationary phase in winter. We also analysed the seasonal expression of the gene encoding the progesterone 5beta-reductase, enzyme producing the required 5beta-configured intermediaries of cardenolides. A fragment of the isolated partial genomic sequence was used as a probe for Northern analysis to study the seasonal gene expression in selected populations. The expression pattern showed increasing levels from February to July and a further reduction in autumn, although harmful climatic conditions seems to induce overexpression of this gene.

Base Sequence↗

Cardenolide genin pattern in Isoplexis plants and shoot cultures.

An HPLC method for the quantitative analysis of cardenolides was developed and applied. The procedure was optimised for analysing small samples (40 mg dw) of plant and tissue culture material. ISOPLEXIS CANARIENSIS plants obtained from seeds accumulated cardenolides to about 20 - 40 micromol g (-1) dw as calculated from the levels of cardenolide genins released after acidic hydrolysis of methanolic extracts. The relative contents of xysmalogenin, digitoxigenin, uzarigenin and canarigenin were 5 - 15 %, 0 - 5 %, 10 - 15 % and 70 - 90 %, respectively. Shoot cultures were initiated from seeds, established as permanent cultures and cultivated on agar-solidified or in liquid medium. Shoot cultures maintained on solid medium contained an average of about 6 micromol cardenolides g (-1) dw. A relatively high proportion of digitoxigenin was found in two-thirds of the shoot cultures examined. The cardenolide content of amphibian shoot cultures averaged to about 1 micromol g (-1) dw. Plants regenerated from shoot cultures and maintained under hydroponic conditions accumulated the same amount of cardenolides as plants collected in the field. No cardenolides could be detected in callus cultures.

Biotechnology↗

The cardenolides of Speirantha convallarioides.

The cardenolide pattern of Speirantha convallarioides has been investigated and compared with Convallaria majalis, a close relative with active cardenolides and used extensively as a cardiac remedy. Five cardenolides were isolated from small amounts (25 g) of plant material by application of an efficient isolation procedure. Their identification was achieved by an easy concept mainly based on detailed analysis of 1H-NMR and DCI-NH3-mass spectral data. Rhodexin A(1) and lokundjoside (2) are the major cardenolides (42% and 31% of the total cardenolides in the plant), while digitoxigenin-glucoside (3), periplorhamnoside (4), and periplogenin-glucoside (5) are minor constituents of the cardenolide complex (3 to 4% each). Similarities in cardenolide patterns (structures of the aglycones, variability of the sugar portion) along with morphological and biogenetical characteristics support the classification of S. convallarioides as belonging in the same tribe Convallarieae as C. majalis within the family Convallariaceae.

Carbohydrate Sequence↗

Cardenolide and Neutral Lipid Biosynthesis from Malonate in Digitalis lanata.

DIGITALIS LANATA plants were grown on water culture in a controlled environment and in the young, growing leaves free sterols (0.335 micromol/g FW), triacylglycerols (0.97 micromol/g FW) and cardenolides (1.82 micromol/g FW) were the major apolar and polar lipids. The cardenolide-containing fraction from these tissues was separated into 26 cardenolides by HPLC. The 5 major components (accounting for 60 % of the occurring glycosides) lanatosides A and C, acetyldigoxin, acetyldigitoxin, and glucoevatromonoside were identified by FABMS. Incorporation experiments with [2- (14)C]-acetate, [2- (14)C]-malonate, [2- (14)C]-mevalonate, and [U- (14)C]-sucrose (absorbed by excised, young, growing leaves) showed the labelling of all the occurring cardenolides after a 3 day incorporation period (as judged by HPLC). Comparing the simultaneous synthesis of labelled sterols and triacylglycerols, malonate could be considered as the most effective precursor in cardenolide synthesis, reaching an incorporation value of 4.1 % in a 4 day incorporation period. A time-course experiment revealed a temporary accumulation of (14)C in glucoevatromonoside, which may play a role as an intermediate in cardenolide production of DIGITALIS LANATA.

Journal Article↗

The inhibition of fibroblast growth factor-2 export by cardenolides implies a novel function for the catalytic subunit of Na+,K+-ATPase.

Basic fibroblast growth factor (FGF-2) is one of a select group of proteins that can exit cells through an alternate, endoplasmic reticulum/Golgi apparatus independent exocytic pathway. This alternate pathway has been termed protein export. In an attempt to better understand this process, we have identified a family of related compounds, "cardenolides," that inhibit FGF-2 export. The cardenolides inhibit FGF-2 export in a time and concentration dependent fashion. Inhibition of FGF-2 export is specific in that the cardenolides have no effect on conventional protein secretion as measured by their inability to block release of the secreted protein human chorionic gonadotropin-alpha. Because cardenolides are known to inhibit ion transport activity mediated by Na+,K+-ATPase, we investigated whether there are functional interactions between FGF-2 and their only known molecular target: the alpha-subunit of Na+, K+-ATPase. Export of FGF-2 from COS-1 cells is selectively inhibited when co-transfected with expression vectors encoding the alpha-subunit and FGF-2. Moreover, antibodies to the alpha-subunit specifically co-immunoprecipitate FGF-2 along with the alpha-subunit while conversely, antibodies to FGF-2 specifically co-immunoprecipitate the alpha-subunit along with FGF-2. Finally, the ion transporting activities of the Na+,K+-ATPase can be uncoupled from protein export. Varying the external concentration of K+ has little effect on export of FGF-2. Taken together, these data: 1) identify a novel activity for cardenolides; 2) suggest a previously unknown role for the alpha-subunit of Na+, K+-ATPase in FGF-2 export; and 3) raise the possibility that the alpha-subunit itself may be an integral component of this alternate exocytic pathway mediating translocation of cytosolic FGF-2 to the cell surface.

Animals↗

[Synthesis and characterization of some cardenolide glucuronides and sulphates (author's transl)].

Cardenolide glucuronides are synthesized in the following way: firstly cardenolide glucosides are prepared by the reaction with acetobromglucose; secondly the hydroxymethyl group of the glucose moiety is oxydized in presence of a platinum catalyst to the carboxyl group of the final glucuronic acid. Glucuronides of the following cardenolides are prepared and described: digoxin, digoxigenin, digitoxin, digitoxigenin-monodigitoxoside, digitoxigenin, and 3-epi-digitoxigenin. Sulphates of digoxigenin, digitoxigenin, and 3-epi-digitoxigenin are prepared by direct reaction of these cardenolides with chlorosulphonic acid in pyridine. The assumed structure of some conjugates has been confirmed by n.m.r. spectroscopy. A high water solubility (6.7-65.1 g/l), a minute chloroform solubility (0.0002-0.0005 g/l), and a low octanol/polar nature of these compounds. Inotropic or toxic cardiac activities of the conjugates are examined on isolated guinea pig papillary muscles and by the Hatcher method on cats. Conjugates with at least one digitoxose show cardioactivities comparable to digoxin or digitoxin. In contrast to that the conjugated genins indicate decreased activities which are at least one-tenth of the potency of the unconjugated glycosides.

Animals↗

Inhibition of Na+,K+-ATPase by the cardenolide 6'-O-(E-4-hydroxycinnamoyl) desglucouzarin.

Among the major cardenolides from the milkweed Asclepias asperula, 6'-O-(E-4-hydroxycinnamoyl) desglucouzarin has not been characterized biochemically. In this study, its binding affinity for a physiological receptor, porcine kidney Na+,K+-ATPase, was found to be lower than the other cardenolides in this plant. The order of affinities from highest to lowest was: uzarigenin (Kd = 1.05 microM) = desglucouzarin (Kd = 0.98 microM) > uzarin (Kd = 4.0 microM) > 6'-O-(E-4-hydroxycinnamoyl) desglucouzarin (Kd = 16 microM). The chemical attachment of the 4-hydroxycinnamoyl group to the 6'-carbon of desglucouzarin significantly inhibits binding. This agrees with predictions that a 5'-methyl group on cardenolides fits the receptor site optimally for the porcine kidney enzyme. The 4-hydroxycinnamic ester was also found to be fluorescent.

Cardenolides↗

Correlation between inhibition of (Na+, K+)-membrane-ATPase and positive inotropic activity of cardenolides in isolated papillary muscles of guinea pig.

Concentrations of 17 cardenolides, cardenolide glucuronides and sulfates producing half-maximal inhibition of (Na+, K+)-membrane-ATPase from different organs and animal species were determined in vitro. In addition the concentrations that increased the contractility of guinea pig isolated papillary muscles to a particular level were investigated. Comparisons between ATPase-inhibiting and positive inotropic cardiac activities showed extensive parallelism: the correlation coefficients after log/log transformation were between 0.92 and 0.97. The same close correlations are found if dissociation constants of cardenolide receptor complexes and concentrations causing 86Rb-uptake inhibition in human erythrocytes are examined. The concentrations necessary for inhibition of (Na+, K+)-membrane-ATPase of the guinea pig heart and the concentrations required to achieve a defined positive inotropic effect in guinea pig papillary muscle showed a log/log correlation coefficient of 0.97 (P less than 0.001). In both tests the potencies covered more than three orders of magnitude. The results support Repke's hypothesis on the digitalis receptor.

Animals↗