Raising antibodies against epoxyscillirosidine, the toxic principle contained in Moraea pallida Bak. (Iridaceae), in rabbits.

Moraea pallida Bak. (yellow tulp) poisoning is the most important plant cardiac glycoside toxicosis in South Africa. The toxic principle, a bufadienolide, is 1α, 2α-epoxyscillirosidine. The aim was to investigate the potential to develop a vaccine against epoxyscillirosidine. Epoxyscillirosidine, proscillaridin and bufalin, were successfully conjugated to hen ovalbumin (OVA), bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH). There was a low immune response following vaccination of adult male New Zealand White rabbits with epoxyscillirosidine-OVA (n = 3) and OVA (n = 3) using Freund's adjuvant in Trial (T) 1. The immune response improved significantly in T2 following doubling of the dose to 0.8 mg/rabbit and changing the adjuvant to Montanide. In T3, the rabbits (n = 15), allocated into 5 equal groups, vaccinated with proscillaridin-BSA, bufalin-BSA, epoxyscillirosidine-KLH, epoxyscillirosidine-BSA and BSA respectively, using Montanide adjuvant, developed antibodies against the administered immunogens, with epoxyscillirosidine-KLH inducing the highest immune response. Proscillaridin and bufalin antibodies cross-reacted with epoxyscillirosidine in an enzyme linked immunosorbent assay. The conjugation methodology will be adjusted in the future to target optimal conjugation efficiency. Additional vaccination will be conducted in search of neutralizing antibodies against the yellow tulp toxin. The cross-reactivity of proscillaridin and bufalin antibodies with epoxyscillirosidine could be studied in future to explore the potential to prevent yellow tulp poisoning.

Synthesis and biological activity of fluorinated analogues of the DAF-12 receptor antagonist 24-hydroxy-4-cholen-3-one.

The DAF-12 receptor is a ligand-activated transcription factor that in its ligand-bound form allows the expression of target genes needed to support the reproductive life cycle of the free-living nematode Caenorhabditis elegans, whereas unbound DAF-12 receptor leads to the developmentally arrested "dauer larvae", specialized for survival and dispersal. The endogenous ligands of the DAF-12 receptor are 3-keto-cholestenoic acids dubbed dafachronic acids. In a previous publication we reported that oxysterols with a shorter side chain (C24) modulate the DAF-12 receptor activity either as partial agonists or, in the case of the C24 alcohol 24-hydroxy-4-cholen-3-one, as an antagonist both in vitro and in vivo. Preliminary structure-activity relationships suggested that this activity profile could be improved with more lipophilic and less acidic functional groups at the end of the side chain. Thus, we have now synthesized two fluorine containing analogues in which the C-24 hydroxyl was replaced by a difluoromethyl group (regarded as a "lipophilic hydroxyl") or a difluoromethylidene group with similar lipophilicity but lacking the hydrogen bond donor capacity. Activity was evaluated in vitro using transactivation cell-based assays and in vivo by the effect on the development of wild-type C. elegans. The 24-difluoromethyl analogue retained the antagonist activity in vitro, being completely devoid of agonist activity and exhibited improved activity in vivo. The difluoromethylidene showed a slight antagonist tendency in vitro (statistically not significant), in the concentration range tested and was weakly active in vivo. None of the compounds were toxic, as treated worms recovered to normal development, when transferred to fresh media without added steroids.

Epoxyscillirosidine Induced Cytotoxicity and Ultrastructural Changes in a Rat Embryonic Cardiomyocyte (H9c2) Cell Line.

Moraea pallida Bak. (yellow tulp) poisoning is the most important cardiac glycoside-induced intoxication in ruminants in South Africa. The toxic principle, 1α, 2α-epoxyscillirosidine, is a bufadienolide. To replace the use of sentient animals in toxicity testing, the aim of this study was to evaluate the cytotoxic effects of epoxyscillirosidine on rat embryonic cardiomyocytes (H9c2 cell line). This in vitro cell model can then be used in future toxin neutralization or toxico-therapy studies. Cell viability, evaluated with the methyl blue thiazol tetrazolium (MTT) assay, indicated that a hormetic dose/concentration response is characterized by a biphasic low dose stimulation and high dose inhibition. Increased cell membrane permeability and leakage, as expected with necrotic cells, were demonstrated with the lactate dehydrogenase (LDH) assay. The LC50 was 382.68, 132.28 and 289.23 µM for 24, 48, and 72 h respectively. Numerous cytoplasmic vacuoles, karyolysis and damage to the cell membrane, indicative of necrosis, were observed at higher doses. Ultra-structural changes suggested that the cause of H9c2 cell death, subsequent to epoxyscillirosidine exposure, is necrosis, which is consistent with myocardial necrosis observed at necropsy. Based on the toxicity observed, and supported by ultra-structural findings, the H9c2 cell line could be a suitable in vitro model to evaluate epoxyscillirosidine neutralization or other therapeutic interventions in the future.

Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism.

The catabolism of sterols in mycobacteria is highly important due to its close relevance in the pathogenesis of pathogenic strains and the biotechnological applications of nonpathogenic strains for steroid synthesis. However, some key metabolic steps remain unknown. In this study, the hsd4A gene from Mycobacterium neoaurum ATCC 25795 was investigated. The encoded protein, Hsd4A, was characterized as a dual-function enzyme, with both 17ß-hydroxysteroid dehydrogenase and ß-hydroxyacyl-CoA dehydrogenase activities in vitro. Using a kshAs-null strain of M. neoaurum ATCC 25795 (NwIB-XII) as a model, Hsd4A was further confirmed to exert dual-function in sterol catabolism in vivo. The deletion of hsd4A in NwIB-XII resulted in the production of 23,24-bisnorcholenic steroids (HBCs), indicating that hsd4A plays a key role in sterol side-chain degradation. Therefore, two competing pathways, the AD and HBC pathways, were proposed for the side-chain degradation. The proposed HBC pathway has great value in illustrating the production mechanism of HBCs in sterol catabolism and in developing HBCs producing strains for industrial application via metabolic engineering. Through the combined modification of hsd4A and other genes, three HBCs producing strains were constructed that resulted in promising productivities of 0.127, 0.109 and 0.074 g/l/h, respectively.

Novel and efficient synthesis of 22-alkynyl-13,24(23)-cyclo-18,21-dinorchol-22-en-20(23)-one analogues.

The efficient synthesis of some 22-alkynyl-13,24(23)-cyclo-18,21-dinorchol-22-en-20(23)-ones was investigated. 22-Iodocyclo-18,21-dinorcholenones were prepared from cyclo-18,21-dinorcholenones using I(2)/DMAP/pyridine system firstly. The cross coupling reaction of 22-iodocyclo-18,21-dinorcholenones and 1-alkynes was carried out efficiently catalyzed by tetrakis(triphenylphosphine) palladium/cuprous iodide in the presence of base diisopropylethylamine. This strategy offered a very straightforward and efficient method for access to conjugated alkynyl cyclo-18,21-dinorcholenones from the cyclo-18,21-dinorcholenones and 1-alkynes in excellent overall yields. Evaluation of the synthesized compounds for cytotoxicity against KB, HeLa, MKN-28 and MCF-7 cell lines showed that the 22-alkynylcyclodinorchoenones possessing hydroxylethyl and hydroxylmethyl mono-substituted side chain at the end of alkynyl group have significantly inhibition activity.

Two ring-A-aromatized bile acids from the marine sponge Sollasella moretonensis.

Two ring-A-aromatized bile acids, 1 and 2, were isolated from the sponge Sollasella moretonensis, collected from the seabed of northern Queensland. Structures were assigned on the basis of extensive 1D and 2D NMR studies, as well as analysis by HRESIMS. Compound 2 has previously been produced synthetically, though this marks its first isolation from a natural source.

Substrate specificity for the 12beta-hydroxylation of bufadienolides by Alternaria alternata.

Hydroxylation is an important route to synthesize more hydrophilic compounds of pharmaceutical significance. Microbial hydroxylation offers advantages over chemical means for its high specificity. In this study, a fungal strain Alternaria alternata AS 3.4578 was found to be able to catalyze the specific 12beta-hydroxylation of a variety of cytotoxic bufadienolides. Cinobufagin and resibufogenin could be completely metabolized by A. alternata to generate their 12beta-hydroxylated products in high yields (>90%) within 8 h of incubation. A. alternata could also convert 3-epi-desacetylcinobufagin into 3-epi-12beta-hydroxyl desacetylcinobufagin as the major product (70% yield). C-3 dehydrogenated products were detected in these reactions in fair yields, while their accumulation was relatively slow. The 12beta-hydroxylation of bufadienolides could be significantly inhibited by the substitution of 1beta-, 5-, or 16alpha-hydroxyl groups, and the 14beta,15beta-epoxy ring appeared to be a necessary structural requirement for the specificity. For the biotransformation of bufalin, a 14beta-OH bufadienolide, this reaction was not specific, and accompanied by 7beta-hydroxylation as a parallel and competing metabolic route. The biotransformation products were identified by comparison with authentic samples or tentatively characterized by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionization-mass spectrometry analyses.

Microbial hydroxylation of bufalin by Cunninghamella blakesleana and Mucor spinosus.

The microbial transformation of a cytotoxic bufadienolide, bufalin (1), was carried out using two strains of filamentous fungi. Cunninghamella blakesleana catalyzed the specific 12alpha-hydroxylation of bufalin and produced 12alpha-hydroxybufalin (2) and 7beta,12alpha-dihydroxybufalin (3) as the major metabolites, together with 7beta-hydroxybufalin (4) and 12beta-hydroxybufalin (5) in low yields. Two minor products were isolated from the culture broth of Mucor spinosus and were identified as 7beta,15alpha-dihydroxybufalin (6) and 5beta,7beta-dihydroxybufalin (7), respectively. Metabolites 2, 3, 6, and 7 are new compounds, and their structures were fully characterized by NMR and MS spectroscopy.

[Progress in biotransformation of triptolides and bufadienolides].

The latest results from our research group in the biotransformation of triptolides and bufadienolides were reviewed. The trends in the development of biotransformation in the future were also briefly discussed.

Apoptosis-mediated selective killing of malignant cells by cardiac steroids: maintenance of cytotoxicity and loss of cardiac activity of chemically modified derivatives.

Cardiac glycosides are commonly used drugs in clinical medicine. We analyzed the cytotoxic effect of six steroids belonging to the bufadienolide family on malignant T lymphoblasts and normal peripheral blood mononuclear cells (PBMC). One compound was a natural bufadienolide glycoside (hellebrin) with cardiac activity. The other five compounds were chemically modified derivatives that did not contain cardioactive groups. We found that these steroids were able to cause time-dependent apoptosis in Jurkat T lymphoblasts, whereas they only minimally affected PBMC. Preferential killing of malignant cells was induced by the natural cardioactive substance hellebrin and by three of the five chemically modified non-cardioactive derivatives. The substances caused mitochondrial transmembrane potential disruption and internucleosomal DNA fragmentation in tumor cells. The cytoplasmic and nuclear events of bufadienolide-induced apoptosis were strongly inhibited in the presence of caspase 8, caspase 9, or caspase 3 inhibitors, as well as in the presence of the broad-spectrum caspase inhibitor Z-VAD-FMK. Overexpression of Bcl-2 significantly protected bufadienolide-treated cells from phosphatidylserine translocation, transmembrane potential disruption, and internucleosomal DNA fragmentation. Our results show that the analyzed bufadienolide derivatives preferentially kill malignant human lymphoblasts by initiating apoptosis via the classical caspase-dependent pathway. Apoptosis-inducing agents specific for tumor cells might be ideal anti-tumor drugs. The therapeutic use of bufadienolides has been hampered by their concomitant cardiac activity. The description of compounds without cardiac activity but with tumor-specific cytotoxicity suggests the potential of using them in cancer therapy.

Conditioned feed aversion as a means to prevent tulp (Homeria pallida) poisoning in cattle.

Conditioned feed aversion was investigated as a means to prevent tulp (Homeria pallida) poisoning in cattle on tulp-infested grazing. Aversion treatment with a combination of epoxyscillirosidin and lithium chloride together with a tulp-hexane extract, which served as identification factor for tulp, resulted in a significantly lower (P < 0.001) proportion of severe tulp poisoning. In a first trial where 21 averted and 21 non-averted control cattle were exposed to a tulp-infested grass pasture, only two of the averted cattle were severely poisoned compared to 13 of the non-averted control cattle. In a second trial, with cattle being exposed to a pure stand of tulp supplemented with maize residues, only two of 21 averted cattle were severely poisoned compared to 14 of 21 non-averted control cattle. Occurrence of mild tulp poisoning, however, did not differ much between averted and non-averted control cattle. The results show that conditioned feed aversion effectively restricted severe poisoning in cattle on tulp-infested grazing.

Bufadienolide and spirostanol glycosides from the rhizomes of helleborusorientalis.

The rhizomes of Helleborus orientalis have been analyzed for the bufadienolide glycoside and spirostanol saponin constituents, resulting in the isolation of a new bufadienolide rhamnoside (1), along with two known bufadienolide glycosides (2 and 3) and five new spirostanol saponins (4-8). The structures of the new compounds were determined on the basis of extensive spectroscopic analysis, including 2D NMR, and the results of hydrolytic cleavage. The isolated compounds were evaluated for their cytotoxic activities against cultured tumor and normal cells.

The effect of three bufadienolide cardiac glycosides on contraction of isolated rat jejunum.

Three cardiac glycosides were screened for pharmacological effects on isolated rat jejunum. The contraction of rat jejunum with epoxyscillirosidin, a non-cumulative bufadienolide, and cotyledoside and tyledoside D, both cumulative neurotoxic bufadienolides were compared with methacholine. The results indicate that all three bufadienolides cause contraction of jejunal smooth muscle. When combined with atropine (1 x 10(-6) M) the response of epoxyscillirosidin and tyledoside D decreased, indicating suppression of a cholinergic response caused by the cardiac glycosides.

[Study on the preparation of venenum bufonis beta-cyclodextrin inclusion complexes].

OBJECTIVE: To study the preparation of Venenum Bufonis beta-cyclodextrin inclusion complexes. METHOD: An optimal condition was established by the uniform design. Under the optimal conditions the Venenum Bufonis beta-cyclodextrin inclusion complexes were prepared with 5 different methods. RESULT: The ball grinding method was superior to other four methods. The bufadienolide inclusion rate of Venenum Bufonis beta-cyclodextrin prepared with ball grinding method was 85.42%. CONCLUSION: Ball grinding method is the best method for the preparation of Venenum Bufonis beta-cyclodextrin inclusion complexes.

Study on the preparation of venenum bufonis beta-cyclodextrin inclusion complexes / 中国中药杂志

<p><b>OBJECTIVE</b>To study the preparation of Venenum Bufonis beta-cyclodextrin inclusion complexes.</p><p><b>METHOD</b>An optimal condition was established by the uniform design. Under the optimal conditions the Venenum Bufonis beta-cyclodextrin inclusion complexes were prepared with 5 different methods.</p><p><b>RESULT</b>The ball grinding method was superior to other four methods. The bufadienolide inclusion rate of Venenum Bufonis beta-cyclodextrin prepared with ball grinding method was 85.42%.</p><p><b>CONCLUSION</b>Ball grinding method is the best method for the preparation of Venenum Bufonis beta-cyclodextrin inclusion complexes.</p>

New insecticidal bufadienolide, bryophyllin C, from Kalanchoe pinnata.

Two insecticidal bufadienolides (1 and 2) were isolated from a methanol extract of the leaves of Kalanchoe pinnata by bioassay-guided fractionation. Compound 1 was identified as known bryophyllin A (bryotoxin C). The structure of new bufadienolide 2, named bryophyllin C, was determined by spectroscopic methods and the chemical transformation of 1. Compounds 1 and 2 showed strong insecticidal activity against third instar larvae of the silkworm (Bombyx mori), their LD50 values being evaluated as 3 and 5 microg/g of diet, respectively.

Limonoids from Azadirachta excelsa.

Activity-directed fractionation of a stem extract of Azadirachta excelsa using KB (human oral epidermoid carcinoma) cells led to the isolation of four meliacin-type limonoids. Two of these constituents were novel, namely, 2,3-dihydronimbolide and 3-deoxymethylnimbidate, and these were purified along with the known compounds, nimbolide and 28-deoxonimbolide. The structures of the new compounds were determined by spectroscopic methods. Nimbolide and 28-deoxonimbolide were broadly cytotoxic when evaluated against a panel of human cancer cell lines, while the two novel compounds were inactive in this regard. The defection of nimbolide and 28-deoxonimbolide as cytotoxic constituents was facilitated by an electrospray LC/MS dereplication procedure.

Ovine cardiac Na,K-ATPase: isolation by means of selective solubilization in Lubrol and the effect of 1 alpha,2 alpha-epoxyscillirosidin on this enzyme.

The inhibition of cardiac Na,K-ATPase by 1 alpha,2 alpha-epoxyscillirosidin is the principal cause of poisoning of cattle by the tulip, Homeria pallida. The ultimate goals of this study were to study the interaction between 1 alpha,2 alpha-epoxyscillirosidin and ovine Na,K-ATPase by means of inhibition and displacement binding studies. Ovine cardiac Na,K-ATPase was isolated in membrane-bound form by means of deoxycholate treatment, high-speed ultracentrifugation, NaI treatment and selective solubilization in Lubrol. The inhibition of ovine cardiac and commercial porcine cerebral cortex Na,K-ATPase by 1 alpha,2 alpha-epoxyscilirosidin and ouabain was studied using a discontinuous Na,K-ATPase assay. The binding of 1 alpha,2 alpha-epoxyscillirosidin, ouabain and digoxin to the above enzymes was compared using a displacement binding assay with [3H] oubain. The Lubrol-solubilized ovine cardiac Na,K-ATPase showed a specific activity of 0.3 U/mg with no ouabain insensitive activity. I50 values of 2.1 x 10(-8) and 2.7 x 10(-8) were obtained for the inhibition of this enzyme by 1 alpha,2 alpha-epoxyscillirosidin and ouabain, respectively. 1 alpha,2 alpha-Epoxyscillirosidin has a much higher KD value (1.5 x 10(-7) M), however, than ouabain (9.5 x 10(-9) M) and digoxin (1.7 x 10(-8) M) in displacement binding studies with [3H]ouabain. 1 alpha,2 alpha-Epoxyscillirosidin is a potent inhibitor of ovine cardiac Na,K-ATPase and is a slightly stronger inhibitor of the enzyme than ouabain. The anomalous result for the displacement of 1 alpha,2 alpha-epoxyscillirosidin from its receptor is either a result of different affinities that K+ has for the enzyme ouabain and enzyme-1 alpha,2 alpha-epoxyscillirosidin complexes or because of different complex stabilities of these complexes.

Bufadienolides and other constituents of Urginea sanguinea.

Fresh bulbs of Urginea sanguinea yielded stigmasterol, phloroglucinol, phloroglucinol 1-beta-D-glucopyranoside (phlorin), scillaren A, a novel compound 5 alpha-4,5-dihydroscillaren A (1), salicylic acid, and 3-hydroxy-4-methylbenzoic acid. The latter two showed weak antibacterial activity. The compounds were identified using spectroscopic techniques such as 1D and 2D NMR, EI-MS and FAB-MS.

Pulse pressure correlates in humans with a proscillaridin A immunoreactive compound.

Endogenous digitalis-like factors in humans are presumably cardenolides and bufadienolides. To test whether bufadienolide-like substances may circulate in human blood, we used antibodies from rabbits against the bufadienolide proscillaridin A to measure the concentration of cross-reacting material in human plasma with an indirect enzyme-linked immunosorbent assay. IgG had an apparent affinity of 2 x 10(-9) mol/L for proscillaridin A. It was specific for bufadienolides and did not cross-react with cardenolides or several steroid hormones. Extraction of human plasma with ethanol and fractionation of this extract over a high-performance liquid chromatographic reverse-phase C18 column with a propanol/isopropanol gradient resulted in the separation of three peaks of increasing hydrophobicity (ED1, ED2, ED3) that inhibited the sodium pump of human red blood cells and cross-reacted with proscillaridin A antibodies. The concentration of the proscillaridin A immunoreactivity ED1 in normotensive subjects had a geometric mean of 0.1 nmol/L, with a dispersion factor of 8.77. ED1 correlated positively in a group of 60 normotensive subjects, 22 patients with hypertension, and 19 patients with chronic renal failure with mean arterial blood pressure (log ED1 [nmol/L] = 0.013 x mm Hg-2.17, r = .25, P < .05), systolic pressure (log ED1 [nmol/L] = 0.010 x mm Hg-2.23, r = .32, P < .01), and pulse pressure (log ED1 [nmol/L] = 0.019 x mm Hg-1.80, r = .38, P < .0001). There was no correlation with other parameters of the donors. We conclude that several substances cross-reacting with proscillaridin A antibodies and inhibiting the sodium pump of human red blood cells circulate in human blood. The level of one of these substances (ED1) correlates with mean arterial and pulse pressures.