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The search for endogenous digitalis has led to the isolation of ouabain as well as several additional cardiotonic steroids of the cardenolide and bufadienolide type from blood, adrenals, and hypothalamus. The concentration of endogenous ouabain is elevated in blood upon increased Na(+) uptake, hypoxia, and physical exercise. Changes in blood levels of ouabain upon physical exercise occur rapidly. Adrenal cortical cells in tissue culture release ouabain upon addition of angiotensin II and epinephrine, and it is thought that ouabain is released from adrenal cortex in vivo. Ouabain levels in blood are elevated in 50% of Caucasians with low-renin hypertension. Infusion over several weeks of low concentrations of ouabain, but not of digoxin, induces hypertension in rats. A digoxin-like compound, which has been isolated from human urine and adrenals, as well various other endogenous cardiac glycosides may counterbalance their actions within a regulatory framework of water and salt metabolism. Marinobufagenin, for instance, whose concentration is increased after cardiac infarction, may show natriuretic properties because it inhibits the alpha1 isoform of Na(+)/K(+)-ATPase, the main sodium pump isoform of the kidney, much better than other sodium pump isoforms. In analogy to other steroid hormones, cardiotonic steroid hormones in blood are bound to a specific cardiac glycoside binding globulin. The discovery of ouabain as a new adrenal hormone affecting Na(+) metabolism and the development of the new ouabain antagonist PST 2238 allows for new possibilities for the therapy of hypertension and congestive heart failure. This will lead in turn to a better understanding of the disease on a physiological and endocrinological level and of the action of ouabain on the cellular level as a signal that is transduced to the plasma membrane as well as to the cell nucleus.

Chemical pollutants can exert various sublethal effects on wildlife, leading to complex fitness consequences. Many animals use defensive chemicals as protection from predators and diseases, yet the effects of chemical contaminants on this important fitness component are poorly known. Understanding such effects is especially relevant for amphibians, the globally most threatened group of vertebrates, because they are particularly vulnerable to chemical pollution. We conducted two experiments to investigate how exposure to glyphosate-based herbicides, the most widespread agrochemicals worldwide, affects the production of bufadienolides, the main compounds of chemical defence in common toads ( Bufo bufo ). In both experiments, herbicide exposure increased the amount of bufadienolides in toad tadpoles. In the laboratory, individuals exposed to 4 mg a.e./L glyphosate throughout their larval development had higher bufadienolide content at metamorphosis than non-exposed tadpoles, whereas exposure for 9 days to the same concentration or to 2 mg a.e./L throughout larval development or for 9 days had no detectable effect. In outdoor mesocosms, tadpoles from 16 populations exhibited elevated bufadienolide content after three-weeks exposure to both concentrations of the herbicide. These results show that pesticide exposure can have unexpected effects on non-target organisms, with potential consequences for the conservation management of toxin-producing species and their predators.

Sea squill (Drimia maritima L.) extracts have been used for centuries for the medical treatment of heart diseases. A procedure for the preparation of Drimia extracts applied for such purposes comprising a fermentation step is described in the German Homoeopathic Pharmacopoeia (GHP). However, little is known about the secondary metabolite profile of such extracts and the fate of these components upon processing and storage. Thus, in the present study sea squill extracts were monitored during fermentation and storage by HPLC-DAD–MSn and GC–MS to characterise and quantitate individual cardiac glycosides and phenolic compounds. For this purpose, a previously established HPLC method for the separation and quantitation of pharmacologically relevant cardiac glycosides (bufadienolides) was validated. Within 12 months of storage, total bufadienolide contents decreased by about 50%, which was attributed to microbial and plant enzyme activities. The metabolisation and degradation rates of individual bufadienolide glycosides significantly differed, which was attributed to differing structures of the aglycones. Further degradation of bufadienolide aglycones was also observed. Besides reactions well known from human metabolism studies, dehydration of individual compounds was monitored. Quantitatively predominating flavonoids were also metabolised throughout the fermentation process. The present study provides valuable information about the profile and stability of individual cardiac glycosides and phenolic compounds in fermented Drimia extracts prepared for medical applications, and expands the knowledge of cardiac glycoside conversion upon microbial fermentation.

A new synthetic analog of bufadienolide, methyl isobryophyllinate A (1), and a known synthetic analog, methyl isobersaldegenate-1,3,5-orthoacetate (2), were obtained by methanolysis of bryophyllin A (3) and bersaldegenin-1,3,5-orthoacetate (5) in basic solution. Structure-insecticidal activity relationship studies revealed both orthoacetate and α-pyrone moieties seemed to be essential structural elements for exhibiting insecticidal activity, whereas oxygenated substituents in the C ring enhanced the insecticidal activity against the third instar larvae of silkworm (Bombyx mori).

The purpose of this study was to develop an alternative submicron emulsion containing three bufadienolides for oral administration and evaluate its preclinical stability, efficacy, and toxicity. The bufadienolide-loaded oral submicron emulsion (BU-OE) was prepared by high-pressure homogenization. The storage stability, in vitro cytotoxicity, in vivo antitumor efficacy, acute toxicity, and long-term toxicity of BU-OE were investigated in detail to evaluate the formulation. The stability study suggested that BU-OE was stable at room temperature and could be stored for at least 18 months at 6±2 °C. The cytotoxicity test revealed that BU-OE had marked cytotoxic activities against cancer cells, but no evident inhibitory effects on normal cells. Likewise, BU-OE exhibited significant antitumor efficacy against Hep G2, HCT-8, and EC9706 cell lines and a slight inhibitory effect on BGC 803 cell line in nude mice, while comparable antitumor activity with fluorouracil injection. The LD50 of BU-OE in mice was 29.4 mg/kg (male) and 22.8 mg/kg (female), respectively. As for the long-term toxicity, BU-OE showed no apparent toxic effects except minor cardiotoxic effects which were reversible. In conclusion, submicron emulsion is a suitable delivery system for oral administration of bufadienolides, with satisfactory stability, superior antitumor efficacy and low toxicity.

The development of bufadienolides as anti-tumor agents is limited due to poor pharmacokinetic properties regarding drug half-lives and toxicity in vivo. These serious factors might be improved by increasing the drug/albumin-binding ratio. This study therefore investigated the relationship between the structural properties of nine bufadienolides and their affinities for human serum albumin (HSA) by a fluorescence spectroscopy-based analysis and molecular docking. Fluorescence quenching data showed that the interaction of each bufadienolide with HSA formed a non-fluorescent complex, while thermodynamic parameters revealed negative ΔS and ΔH values, corresponding to changes in enthalpy and entropy, respectively. The structural differences between the various bufadienolides markedly influenced their binding affinity for HSA. With the exception of a C = O bond at the C12 position that decreased the binding affinity for HSA, other polar groups tended to increase the affinity, especially a hydroxyl (OH) group at assorted bufadienolide sites. The rank order of binding affinities for drugs with tri-hydroxyl groups was as follows: 11-OH > 5-OH > 16-OH; in addition, 16-acetoxy (OAc), 10-aldehyde and 14-epoxy constituents notably enhanced the binding affinity. Among these groups, 11-OH and 16-acetyl were especially important for a seamless interaction between the bufadienolides and HSA. Furthermore, molecular docking analysis revealed that either an 11-OH or a 16-OAc group spatially close to a five-membered lactone ring significantly facilitated the anchoring of these compounds within site I of the HSA pocket via hydrogen bonding (H-bonding) with Tyr150 or Lys199, respectively. In summary, bufadienolide structure strongly affects binding with HSA, and 11-OH or 16-OAc groups improve the drug association with key amino acid residues. This information is valuable for the prospective development of bufadienolides with improved pharmacological profiles as novel anti-tumor drugs.

A high yielding route to bufadienolide type steroids using a novel Suzuki coupling reaction between a range of steroid vinyl triflates and 2-pyrone-5-boronate 11 is presented.

Three bufadienolides, hellebortin A (5-[beta-D-glucopyranosyloxy]-10,14,16-trihydroxy-19-nor-[5beta,10beta,14beta,16beta]-bufa-3,20,22-trienolide [1]), hellebortin B (5-[beta-D-glucopyranosyloxy]-3,4-epoxy-14-hydroxy-19-oxo-bufa-20,22-dienolide [2]) and hellebortin C (5-[beta-D-glucopyranosyloxy]-3,4-epoxy-10,14-dihydroxy-19-nor-bufa-20,22-dienolide [3]), together with 20-hydroxyecdysone 3-O-beta-D-glucoside (4) and 20-hydroxyecdysone (5) have been isolated by bioassay- and RIA-directed HPLC analyses of a methanol extract of the seeds of Helleborus torquatus. The structure and relative stereochemistry of the novel bufadienolide hellebortin A (1) and the structures of hellebortin B (2) and hellebortin C (3) were determined unambiguously by comprehensive analyses of their 1D and 2D NMR data. These five compounds are isolated from Hellborus torquatus for the first time. The biological activities of compound 1, 4 and 5 as ecdysteroid agonists and antagonists have been assessed.

Thirty three compounds were obtained from the bulbs of Urginea maritima (Liliaceae). The compounds were identified by means of fast atom bombardment mass spectrometry (FAB-MS), nuclear magnetic resonance (1H-NMR), (13C-NMR), nuclear overhauser effects (NOE) and two dimensional (2D) NMR. Ten of them were new natural compounds. Nine were bufadienolides and only one was lignan in these compounds.

To determine plasma levels of the endogenous bufodienolide Na+/K+ ATPase inhibitor, marinobufagenin-like factor (MBG), in normotensive pregnancy and in preeclampsia, to compare changes of MBG with that of ouabain-like compound (OLC), and to characterize the purified MBG immunoreactive factor from preeclamptic plasma.Consecutive sample study. The levels of MBG and OLC compounds were measured in extracted plasma by solid phase fluoroimmunoassays. MBG and ouabain immunoreactive materials were partially purified from preeclamptic plasma via reverse-phase high-performance liquid chromatography (HPLC) and studied for their ability to cross react with MBG and ouabain antibodies, and to inhibit the Na+/K+ ATPase from human mesenteric arteries. Vasoconstrictor effect of authentic MBG was studied in isolated rings of human umbilical arteries.In 11 nonpregnant control individuals, plasma concentrations of MBG and OLC were 0.190+/-0.04 nmol/l and 0.297+/-0.037 nmol/l, respectively. In the third trimester of noncomplicated pregnancy (n = 6), plasma MBG increased (0.625+/-0.067 nmol/l, P0.05), and OLC did not (0.32+/-0.07 nmol/l). In 15 patients with preeclampsia, plasma levels of both MBG and OLC increased dramatically (2.63+/-0.10 nmol/l and 0.697+/-0.16 nmol/l, respectively, P0.01 versus both control groups). When fractionated by reverse phase HPLC, OLC was eluted by 18% acetonitrile, and MBG by 48% acetonitrile. Serially diluted samples of MBG and OLC immunoreactive materials from HPLC fractions reacted with MBG and ouabain antibody in solid phase immunoassay in a concentration dependent fashion. Authentic MBG caused contractile responses of isolated rings of human mesenteric arteries in a concentration-dependent manner. Similarly to the authentic MBG, HPLC purified MBG immunoreactive material from preeclamptic plasma inhibited Na+/K+ ATPase purified from human mesenteric artery.Our observations demonstrate the coexistence of two endogenous cardiotonic steroids in preeclamptic plasma, a more polar OLC and a less polar MBG-like compound. Substantial increases in plasma OLC and MBG immunoreactivity in preeclampsia, along with the vasoconstrictor properties of authentic MBG and Na+,K+ ATPase inhibitory activity of human MBG immunoreactive factor, suggest, that in preeclampsia, plasma concentrations of MBG are enough to substantially inhibit the sodium pump in cardiovascular tissues, and are in accordance with the views attributing endogenous digitalis-like factors a pathogenic role in the preeclamptic hypertension.

Chan Su, a traditional Chinese medication, and Love Stone, a topical aphrodisiac, are both made from dried venom of the toad bufo bufo gargarizans and contain bufalin, cinobufotalin, cinobufagin, and other cardioactive steroids of the bufadienolide class. Deaths have occurred following ingestion of these products and the clinical course resembles digoxin toxicity. The purpose of this study was to determine the efficacy of digoxin specific Fab fragments in treating Chan Su poisoning. An ethanolic extract was prepared from Chan Su. Digoxin specific Fab fragments were reconstituted in normal saline to a concentration of 80 mg/ml. An approximate LD90 dose was determined in preliminary experiments. Mice were then randomly divided into a treatment group of 15 mice and a control group of 30 mice. The treatment group was pretreated with 20 ml/kg of digoxin specific Fab fragment solution by intraperitoneal injection at t = 0, followed by 10 ml/kg of digoxin specific Fab fragments intraperitoneal at t = 30 min. The control group was pretreated with equal volumes of intraperitoneal normal saline at the same times. Immediately following the 30 min injection, both groups were given the estimated LD90 dose of Chan Su extract by subcutaneous injection. An endpoint of survival at 6 h was chosen after preliminary results showed that all deaths occurred in the first 4 h. All 30 of the control mice had seizures followed by death compared to 11 seizures and 7deaths in the 15 treatment mice. These results were statistically significant by Fisher's exact test (p = 0.00003 for mortality and p = 0.009 for seizures). Digoxin specific Fab fragments are effective in the treatment of Chan Su poisoning in mice and may be effective for poisoning by other cardioactive steroids of the bufadienolide class.

We have examined the column chromatography using hydrophobic gel carriers, Sephadex LH-20 and HP-Cellulofine, for separation of ten toad poison bufadienolides: resibufogenin, cinobufagin, bufalin, bufotalin, cinobufotalin, telocinobufagin, desacetyl-cinobufagin, desacetyl-bufotalin, desacetyl-cinobu-fotalin, and gamabufotalin (Figure 1). As a result, the use of developing solvents such as n-hexane/CH2Cl2/MeOH(4:5:1), n-hexane/CHCl3/MeOH(4:5:1), and n-hexane/toluene/ MeOH (3:2:1) provided the effective separation of bufadienolides. For separation, we have established the flow speed of developing solvents and the relationship of separation with the diameter and length of column, in addition to the comparison of separation by Sephadex LH-20 and HP-Cellulofine and the relationship with silica gel thin-layer chromatography. Also, we have tested the separability of hydrophobic gel by repeated use and the recovery of bufadienolides on hydrophobic gel column by repeated use. 10.1081/JLC-100101814-F0001 Figure 1. ...

Cunninghamella elegans has been employed for the biotransformation of cinobufagin (1) to afford one metabolites. The structure of the transformation product has been characterized as 7β,12β-dihydroxylcinobufagin (2). Product 2 is a new compound. In vitro cytotoxic activities of the biotransformation product and the substrate-cinobufagin have been assayed against HeLa; they all showed cytotoxic activities.

Introduction Marinobufagenin (MBG) is an endogenous bufadienolide cardiac inotrope which is demonstrating growing interest in the early diagnosis of volume expansion-mediated hypertensive states such as preeclampsia (PE) and end-stage renal disease hypertension. Mammalian MBG is an inhibitor of the α 1 isoform of Na + , K + -ATPase with vasoconstrictive and cardiotonic properties, resulting in hypertension and natriuresis. Elevated endogenous MBG levels have been described in pregnant mammals and especially in preeclamptic patients [1,2,3]. The rise of endogenous MBG appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential target in the biomarker panel for PE. A sensitive and accurate analytical method is needed to assess MBG in as lower level as possible in plasma. Currently, only marinobufagenin-like material has been found in humans using two published quantification methods based each on immunoassays [4,5]. These techniques suffer from a lack of specificity due to cross-reactivity and tend to exhibit high variability at low concentrations [6]. Objective Our aim is to develop a MBG assay using a more specific and easy to access technique, such as LC–MS/MS. An algorithm dealing with the MBG plasma levels might be established by clinicians in the future, in order to predict, in combination with other clinical and biological markers, the risk for preeclampsia in pregnant women. Methods As the major source for MBG is located in the parotid glands of the Bufo Marinus toad, we developed a purification method from toad venom in order to get pure MBG standard. A pre-extraction procedure was elaborated to concentrate and clean the plasma sample prior to its analysis. A LC–MS based assay designed to determine MBG in human plasma is being optimized, giving us the opportunity to investigate MBG in non-pregnant healthy volunteers plasma as well as in early pregnant women plasma. Results and discussion Pure MBG has been successfully extracted from the B. Marinus toad venom and the identity of the compound has been confirmed. An extraction procedure for MBG from plasma has been set up by use of solid phase extraction cartridges. Preliminary results allowed us to authenticate the presence of MBG by LC–MS/MS in non-pregnant women as well as in early pregnancy. Further optimization and validation of the LC–MS assay are needed to quantify MBG plasma levels. However, these pioneering observations, are giving the clinicians a promising perspective for early preeclampsia risk assessment in pregnant women.

Naturally occurring bufadienolides, which were isolated from both animal and plant sources and structurally elucidated in the period from 1967–1995, are reviewed and compiled.

Bufonid frogs of the genus Atelopus contain two classes of skin toxins, namely the steroidal bufadienolides and the water-soluble tetrodotoxins. Frogs of the Panamanian species Atelopus varius have now been raised in captivity and levels in skin extracts of bufadienolides and of tetrodotoxin-like compounds assessed, using inhibition of [3H]ouabain binding and inhibition of [3H]saxitoxin binding, respectively. Levels of ouabain equivalents, corresponding to bufadienolides, were comparable to those found in wild-caught frogs from the same population in Panama, while tetrodotoxin-like activity was undetectable. The results strongly implicate environmental factors, perhaps symbiotic microorganisms, in the genesis of tetrodotoxins in the skin of frogs of the genus Atelopus, while indicating that the frog itself produces the skin bufadienolides.

Two new cardenolides, kalantubolide A (1) and kalantubolide B (2), and two bufadienolide glycosides, kalantuboside A (3) and kalantuboside B (4), as well as eleven known compounds were isolated and characterized from the EtOH extract of Kalanchoe tubiflora. The structures of compounds were assigned based on 1D and 2D NMR spectroscopic analyses including HMQC, HMBC, and NOESY. Biological evaluation indicated that cardenolides (1-2) and bufadienolide glycosides (3-7) showed strong cytotoxicity against four human tumor cell lines (A549, Cal-27, A2058, and HL-60) with IC50 values ranging from 0.01 µM to 10.66 µM. Cardenolides (1-2) also displayed significant cytotoxicity toward HL-60 tumor cell line. In addition, compounds 3, 4, 5, 6, and 7 blocked the cell cycle in the G2/M-phase and induced apoptosis in HL-60 cells.

The in vitro growth inhibitory effects of 27 bufadienolides and eight degradation products, with two cardenolides (ouabain and digoxin) chosen as reference compounds, were analyzed by means of an MTT colorimetric assay in six human and two mouse cancer cell lines. A structure−activity analysis was then performed to highlight the most important substituents relating to the in vitro growth inhibitory activity of bufadienolides in cancer cells. Thus, the current study revealed that various bufadienolides, including gamabufotalin rhamnoside (1a), bufotalin (2a), and hellebrin (3a), displayed higher growth inhibitory activities for various human cancer cell lines when compared to ouabain and digoxin. Gamabufotalin rhamnoside (1a) was the only compound that displayed growth inhibitory effects of

Three major groups of endogenous digitalis-like substances (EDLS) have been identified in the plasma of the toad, Bufo marinus. One group of compounds, present in fresh plasma, is composed of chromatographically homogeneous polar conjugates, principally bufadienolide 3-sulfates, which exhibit relatively weak Na(+)-K(+)-adenosinetriphosphatase (ATPase) inhibitory activity. A second and larger group of compounds, also found in fresh plasma, includes chromatographically heterogeneous conjugates, which are effective inhibitors of Na(+)-K(+)-ATPase; these compounds possess properties similar to those of bufotoxins. The third group of EDLS consists of free unconjugated bufadienolides, which are also effective Na(+)-K(+)-ATPase inhibitors. These unconjugated bufadienolides are present in relatively low concentrations in fresh toad plasma, but appreciable quantities are enzymatically generated from conjugates (believed to consist principally of bufotoxins) during the in vitro incubation of plasma. We suggest that the extent to which circulating polar EDLS are enzymatically deconjugated in vivo may be important in the regulation of the digitalis-sensitive Na(+)-K(+)-ATPase of toad brain, the only known digitalis-sensitive Na(+)-K(+)-ATPase in the toad.

Abstract 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 2×10 −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 (ED 1 , ED 2 , ED 3 ) that inhibited the sodium pump of human red blood cells and cross-reacted with proscillaridin A antibodies. The concentration of the proscillaridin A immunoreactivity ED 1 in normotensive subjects had a geometric mean of 0.1 nmol/L, with a dispersion factor of 8.77. ED 1 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 ED 1 [nmol/L]=0.013×mm Hg−2.17, r =.25, P 1 [nmol/L]=0.010×mm Hg−2.23, r =.32, P 1 [nmol/L]=0.019×mm Hg−1.80, r =.38, P 1 ) correlates with mean arterial and pulse pressures.

Achievements in the synthestic study on cardiotonic steroids, pyranonaphthoquinone antibiotics, and tetronic acid ionophore antibiotics from author's laboratory are reviewed.

Derivatives of bufogenin isolated from the skin of the Chinese toad, Bufo bufo gargarizans Cantor ("Ch'an Su"), and several semisynthetic derivatives of 20beta,21beta-epoxy-resibufogenin (13) have been evaluated for interleukin-6 (IL-6) antagonistic activity due to their growth-inhibitory activities on IL-6-dependent MH-60 cells. Among the naturally derived compounds (1-17), 20S,21-epoxy-resibufogenin formate (1) showed potent inhibitory activity on the IL-6-dependent growth of MH-60 cells. Epoxide groups at both the C-14, C-15 and C-20, C-21 positions are required to exhibit this type of activity. Compounds acetylated at the C-16 position (7 and 9-11) showed a loss of activity. An oxo group at the C-3 position (8, 14, and 15) resulted in cytotoxicity for both cell lines. Stereochemistry is important for selectivity on suppression of IL-6 activity. Among the semisynthetic derivatives (18-25) of 13, compound 19, with an acetyl group introduced at the C-3 position in comparison to 13, demonstrated considerable growth inhibition of IL-6-dependent MH-60 cells.

Introduction: Marinobufagenin (MBG), an endogenous bufadienolide cardiac inotrope, is responsible for vasoconstriction and natriuresis [1,2]. Growing interest by its implication in volume expansion...

Objective To kinds of establish a HPLC method for determining contents of indole alkaloids and bufadienolides contained in toad medicines, and analyze two kinds of components contained in toad venom, toad skin and toad periostracum. Method As for alkaloids, Nucleosil C18 column was adopted with acetonitrile and water containing 0.5% potassium dihydrogen phosphate (6: 94, adjust pH to 3.2 with phosphate acid) as the mobile phase. The flow rate was 0.8 mL x min(-1), the detection wavelength was 275 nm, and the column temperature was 30 degrees C. As for bufadienolides, Alltima C18 column was adopted with acetonitrile and water containing 0.3% acetic acid (B) as the mobile phase. The gradient process was as follows: a linear gradient from 28% to 54% acetonitrile in the first 15 min, then kept at 54% for additional 20 min. The flow rate was 0.6 mL x min(-1), the detection wavelength was 296 nm, and the column temperature was 30 degrees C. Result The linear ranges were 0.079 6-0.796 microg for serotonin, 0.097 2-1.945 microg for N-methylserotonin, 0.074 4-0.744 microg for N,N-dimethylserotonin, 0.103-2.05 microg for N,N,N-trimethylserotonin, and 0.067 2-0.672 microg for bufothionine, respectively. The average recoveries of serotonin and N-methylserotonin were 98.6% and 91.3%, respectively. The linear ranges of gamabufotalin, bufotalin, bufalin, cinobufagin and resibufogenin were 0.004 83-0.614, 0.007 9-1.006, 0.007 95-1.016, 0.009 7-1.24 and 0.009 6-1.22 microg, respectively, and their average recoveries were 101.6%, 102.5%, 101.0%, 99.1% and 98.9%, respectively. Conclusion Toad venom has the highest contents of indole alkaloids and bufadienolides, followed by toad skin, and toad periostracum showed the lowest contents and even no detection result.

Molecular docking studies have shown that Δ8,14-anhydrobufalin (1) exhibited more potent binding affinity on androgen receptor (AR) than Δ14,15-anhydrobufalin (2) and bufalin (3). To validate the docking results, compounds 1 and 2 were synthesized. The AR competitive binding assay indicated that the IC50 values of 1–3 were 1.9, >50 and >50 μM (relative binding affinity), respectively, which confirmed that our theoretical binding mode was reliable and predictable. Furthermore, compound 1 was found to show more potent inhibitory activity against the androgen dependent LNCaP cancer cells than the androgen independent PC3 cancer cells, but exhibited less inhibition on the Na+/K+ ATPase as compared with the parent compound 3. To the best of our knowledge, compound 1 represented the first AR antagonist derived from bufadienolide discovered through a series of combined approaches of molecular docking and actual experimental validation.

Ethnopharmacological relevance Medicinal plants from the Sinai desert are widely used in traditional Bedouin medicine to treat a range of conditions including, cancers, and may thus be useful sources of novel anti-tumor compounds. Information on plants used in this way was obtained through collaboration with Bedouin herbalists. Aim of the study To document the traditional uses of 61 species from 29 families of Egyptian medicinal plants and to investigate their biological activity using a cytotoxicity assay. Material and methods MeOH extracts of the 61 plant species investigated were dissolved in 10% DMSO and their cytotoxic activity was evaluated. The extracts were tested in duplicate on three separate occasions at three different concentrations (1, 10 and 100 μg/ml) against human lymphoma U-937 GTB. The most active extract was subjected to bioassay-guided fractionation using HPLC and LC/ESI–MS to isolate and identify its active components. Results and discussion The most potent extracts were those from Asclepias sinaica, Urginea maritima, Nerium oleander and Catharanthus roseus, followed by those from Cichorium endivia, Pulicaria undulate and Melia azedarach. Literature reports indicate that several of these plants produce cardiac glycosides. Bioassay-guided fractionation of alcoholic U. maritima extracts led to the isolation of a bioactive bufadienolide that was subsequently shown to be proscillaridin A, as determined by 1D and 2D NMR spectroscopy. This result demonstrates the value of plants used in traditional medicine as sources of medicinally interesting cytotoxic compounds.

The active components from the extracts of Digitalis, cardiotonic steroid glycosides, have been ingested by humans for more than 200 years as a medicinal therapy for heart failure and abnormal heart rhythms. The positive inotropic activity of the cardiotonic steroids that mediates clinically useful physiological effects in patients has been attributed largely to a high affinity inhibitory interaction with the extracellular surface of the membrane-bound sodium pump (Na(+)/K(+)-ATPase). However, previously unrecognized intracellular signaling pathways continue to be uncovered. This Review examines both partial and de novo synthetic approaches to the medicinally important and structurally captivating cardenolide and bufadienolide steroid families, with an emphasis on the stereocontrolled construction of the pharmacophoric aglycone (genin) framework.

Drug toxicity may occur due to dangerous drug combination. We aimed to investigate the influence of verapamil (a P-gp inhibitor)--bufadienolides interaction on cardiotoxicity and bufadienolide uptake by the isolated heart. The study was performed in Langendorff isolated perfused guinea-pig hearts by bufadienolides infusion in the absence and presence of verapamil (250, 500ng/ml). Arrhythmia parameters were evaluated by ECG and the content of bufadienolides in heart were measured by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS). In the present of verapamil, the wide QRS duration and lightly rapid heart rate (HR) were markedly reduced in the early stage of bufadienolide intoxication. However, the ECG changes characterized by prolonged P-R interval, and slow heart rate and low QRS amplitude in the late stage of bufadienolide intoxication were significantly enhanced. Furthermore, the contents of a variety of bufadienolide compounds in the verapamil+bufadienolide group were significantly higher when cardiac arrest occurred. Although verapamil reduced the bufadienolide-induced ventricular arrhythmias, verapamil worsened heart block and lethal bradycardia of bufadienolides partly via increasing the uptake of bufadienolides in heart tissue, which could compromise the protective effects of verapamil against bufadienolide intoxication. These results suggested that the verapamil may produce dangerous interactions with drugs containing bufadienolides.

Human leukemia K562 cell differentiation induction by naturally occurring bufadienolides purified from the Chinese drug Senso and synthetic bufalin derivatives was examined by a nitro blue tetrazolium reduction assay. Bufalin showed the strongest activity among all the bufadienolides tested in this study. The degree of the induction of nitro blue diformazan positive cells by the bufadienolides correlated well with their inhibitory activities against Na+,K(+)-ATPase prepared from K562 cells in vitro. N+,K(+)-ATPases from a variant K562 clone (ouabain resistant, OuaR) and murine leukemia cell line M1-T22, which were insensitive to the bufadienolides in terms of growth inhibition and cell differentiation, appeared to be refractory to bufalin in vitro. A binding study of 3H-bufalin and 3H-ouabain revealed that saturated levels of both ligands associated with K562 cells were virtually similar; however, affinity of 3H-bufalin was considerably higher than 3H-ouabain. The saturated level of 3H-bufalin observed in the OuaR cells was approximately half of that observed in K562 cells without a change in its affinity. Association of 3H-bufalin with K562 cells was completely blocked by pretreatment of the cells with cold ouabain at concentrations saturating the binding sites. These results suggest that bufalin acts on the cells by binding to sites on the cell membrane which also bind ouabain. It is thus proposed that N+,K(+)-ATPase inhibition is closely related to the initiation process in the induction of K562 cell differentiation induced by bufalin.

Red coloured bulbs of plants of the URGINEA MARITIMA aggregate from the northern and southern Mediterranean area were shown to exhibit different bufadienolide compositions. Therefore, the bufadienolide complex of "red squill" samples from Tunesia and from Sardegna was investigated. Twelve bufadienolides were isolated from bulbs of tetraploid squill from Tunesia, sixteen from tetraploid samples from Sardegna. The Compounds were identified by means of FAB-MS, (1)H-NMR, and (13)C-NMR studies or comparison with authentic substances. Both of the samples contained scilliroside ( 14), scillarenin 3- O-beta- D-glucoside ( 2), proscillaridin A (1), scilliphaeosidin 3- O-beta- D-glucoside ( 5), scilliglaucoside ( 17), scilliphaeoside ( 4), and 12- EPI-scilliphaeoside ( 7); the bulbs from Tunesia contained additionally glucoscilliphaeoside ( 6) and the four new bufadienolides 12- EPI-glucoscilliphaeoside ( 10), 12beta-hydroxyscilliglaucosidin 3- O-beta- D-glucoside ( 21), 12- EPI-scilliphaeosidin 3- O-beta- D-glucoside ( 8), and 12- EPI-scilliphaeosidin 3- O-alpha- L-rhamnosido-alpha- L-rhamnoside ( 9). In the bulbs from Sardegna scularen A ( 3), gamabufotalin 3- O-alpha- L-rhamnoside ( 20), scilliglaucoside ( 17), scillirubrosidin 3- O-alpha- L-rhamnoside ( 11), scillirubroside ( 12), 12beta-hydroxyscilliroside ( 15), 5alpha-4,5-dihydroscillirosidin 3- O-alpha- L-thevetosido-beta- D-glucoside ( 19), and deacetyl-scilliroside ( 8) besides the new Compounds 10-carb-oxy-5beta,14beta-dihydroxybufa-3,20,22-trienolide 5- O-beta- D-glucoside ( 18) and scilliglaucogenin ( 16) were found in addition. Not only quantitative but even qualitative differences in the bufadienolide pattern between the samples of the northern and the southern Mediterranean area were proved. By now it is not clear whether the investigated samples belong to different chemodemes of URGINEA NUMIDICA or are two different species of the URGINEA MARITIMA aggregate.

The effects of a bufadienolide isolated from toad venom, arenobufagin, a potent Na+/K+ pump inhibitor, were studied in single guinea-pig ventricular cells in the whole-cell patch-clamp configuration. Arenobufagin (50 microM) applied extracellularly decreased the amplitude of the delayed rectifier K+ current (IdK) by 30% without affecting the gating kinetics. The L-type Ca2+ current was also depressed, but to a lesser extent. The inward rectifier K+ current was hardly affected. Ouabain and the internal dialysis of cells with the solution containing 20 mM Na+ depressed IdK in a similar way as arenobufagin. On the other hand, arenobufagin also depressed IdK when the Na+/K+ pump was already inhibited in the K(+)-free Tyrode solution. Therefore, both a direct effect on the channel and an indirect effect through the inhibition of the Na+/K+ pump may be involved in the depression of IdK by arenobufagin.

The structure of a new bufadienolide glycoside, cinobufagin 3-O-β-D-glucoside, was determined by extensive 1D and 2D NMR techniques (1H, 13C, DEPT, COSY, HSQC, g-HMBC and NOESY). The complete assignments of the 13C and 1H spectral data were also carried out. Copyright © 2002 John Wiley & Sons, Ltd.

Bufadienolides, known ligands of the sodium pump, have been shown to inhibit the proliferation of several cancer cell types. However, their development to date as anticancer agents has been impaired by a narrow therapeutic margin resulting from their potential to induce cardiotoxicity. In the present study, we examined the effects of bilirubin, an endogenous antioxidant, on the cardiotoxicity of bufadienolides (derived from toad venom) in guinea-pigs. The results showed that bufadienolides (8 mg/kg) caused ventricular arrhythmias, conduction block, cardiac dysfunction and death in guinea-pigs. Pretreatment with bilirubin (75 and 150 mg/kg) significantly prevented bufadienolide-induced premature ventricular complexes, ventricular tachycardia, ventricular fibrillation and death. Bilirubin also markedly improved the inhibition of cardiac contraction in bufadienolide-treated guinea-pigs as evidenced by increases in left ventricular systolic pressure and decreases in left ventricular diastolic pressure in vivo. Furthermore, bilirubin significantly reduced the intracellular sodium content ([Na(+)]( i )) in ex vivo bufadienolide-stimulated guinea-pig ventricular myocytes loaded with the sodium indicator Sodium Green. An antitumor study showed that bilirubin did not compromise the ability of bufadienolides to inhibit gastric cancer cell MGC-803 proliferation. These results suggested that bilirubin can attenuate bufadienolide-induced arrhythmias and cardiac dysfunction in guinea-pigs by reducing elevated [Na(+)]( i ) and may improve bufadienolide therapeutic index in cancer treatment.

Covering: 1998 to July 2010 Since 1998, a great number of bufadienolides have been reported from plants, animals and plant cell suspensions, as well as from fungi and bacteria cultures. This review summarizes both new bufadienolides and those obtained from new sources, together with data regarding the antitumor activity of bufadienolide derivatives.

Nine furostanol glycosides, namely caucasicosides E–M, were isolated from the MeOH extract of the leaves of Helleborus caucasicus, along with 11 known compounds including nine furostanol glycosides, a bufadienolide and an ecdysteroid. Their structures were established by the extensive use of 1D and 2D NMR experiments along with ESIMSn analyses. The steroidal composition of leaves of H. caucasicus shows as particular feature the occurrence of steroidal compounds belonging to the 5β series, unusual for Helleborus species, and in particular, caucasicosides F–H are based on a 5β-polyhydroxylated steroidal aglycon never reported before.

To study the chemical composition of the skin of Bufo bufo gargarizans, various chromatographic methods were used in the isolation procedures and the structures of isolated compounds were determined based on NMR and MS analysis. As a result, two new compounds were isolated from its ethanolic extract and characterized as N-[2-hydroxy-2-(4-hydroxyphenyl)ethyl]-N-methylurea (1) and 19-oxocinobufotalin 3-adipoylarginine ester (2), together with 11 known compounds. Isolated bufadienolides showed significant inhibition effect against human hepatocellular carcinoma cell line SMMC-7721 in vitro.

The death receptor ligand TRAIL is considered a promising candidate for cancer therapy because of its preferential toxicity to malignant cells. However its efficacy has been challenged by a number of resistance mechanisms. Therefore, agents that can overcome the resistance to enhance therapeutic efficacy of TRAIL are needed. In the current study, we found that bufalin, bufotalin and gamabufotalin, key members of bufadienolides isolated from a traditional Chinese medicine ChanSu, significantly potentiated human breast cancer cells with different status of ER-alpha to apoptosis induction of TRAIL, as evidenced by enhanced Annexin V/FITC positive cells (apoptotic cells), cytoplasmic histone-associated-DNA-fragments, membrane permeability transition (MPT), caspases activation and PARP cleavage. Further mechanistic investigation demonstrated that bufalin was able to significantly decrease Mcl-1 expression and modestly decrease Bcl-XL expression level. Down-regulations of these anti-apoptotic proteins were well correlated with inhibition of transcription factor STAT3 activation. The important consequence of down-regulation Mcl-1 in the enhancement action by combining bufalin with TRAIL was confirmed by either knockdown or overexpression of Mcl-1 approach. Our findings for the first time provided strong evidences that bufadienolide compounds have excellent potential to be developed as a novel class of sensitizers of TRAIL.

The effects of arenobufagin, a bufadienolide isolated from toad venom, on the Na + /K + pump were investigated by recording the whole-cell current of dissociated cardiac myocytes. Arenobufagin blocked the Na + /K + pump current in a dose-dependent manner with a half-maximal concentration of 0.29 μM and a Hill coefficient of 1.1. Arenobufagin is 3.5-fold more potent than ouabain.

A naturally occurring bufadienolide, proscillaridin 1a, and its aglycone, scillarenin 1b, were subjected to photolysis in MeOH with a high-pressure mercury are lamp to afford novel 14β, 21-epoxy-21-methoxycholadienes 2a–4a and 2b–4b and 14β,21-epoxy -22-methoxycholadienes 5a, 5b, 6a and 6b, along with 14β,21-epoxycholatrienes 7a and 7b. These structures were elucidated from spectroscopical evidence including NOE experiments.

Cardioactive glycosides, like digoxin, ouabain and related compounds, are drugs that inhibit Na(+)/K(+)-ATPase and have a strong inotropic effect on heart: they cause the Na(+)/Ca(2+) exchanger to extrude Na+ in exchange with Ca(2+) and therefore increase the [Ca(2+)](i) concentration. For this reason, some of these drugs are currently used in the treatment of congestive heart failure and cardiac arrhythmias. Recently it has been discovered that cardiac glycosides exert pleiotropic effects on many aspects of cell metabolism. Na(+)/K(+)-ATPase is not the exclusive target, as they affect the cell response to hypoxia, modulate several signaling pathways involved in cell death and proliferation, regulate the transcription of different genes and modify the pharmacokinetics of other drugs, by altering the expression and activity of drug-metabolizing enzymes. Some of these effects are related to the steroid structure of glycosides, a property which also makes them fine modulators of the synthesis of cholesterol and steroid hormones. Moreover, new endogenously synthesized glycosides have been discovered in the last years: these molecules are involved in the balance of salt and in the control of blood pressure. This review will focus on the recent studies which have demonstrated that exogenous and endogenous glycosides, besides playing a role as inotropic agents, are also important in the pathogenesis and therapy of different human diseases, such as stroke, diabetes, neurological diseases and cancer.

A naturally occurring bufadienolide, proscillaridin 1a, and its aglycone, scillarenin 1b, were subjected to photolysis in MeOH with a high-pressure mercury are lamp to afford novel 14β, 21-epoxy-21-methoxycholadienes 2a–4a and 2b–4b and 14β,21-epoxy -22-methoxycholadienes 5a, 5b, 6a and 6b, along with 14β,21-epoxycholatrienes 7a and 7b. These structures were elucidated from spectroscopical evidence including NOE experiments.

A high yielding route to bufadienolide type steroids using a novel Suzuki coupling reaction between a range of steroid vinyl triflates and 2-pyrone-5-boronate 11 is presented.

Toad venom, namely, "Chansu" in China, has been widely used for the treatment of heart failure, sores, pains, and various cancers. Upon LC-MS analysis of the venom from Bufo melanosticus collected in Indonesia, new bufadienolide sulfates were identified. For a complete characterization, the MeOH extract of the toad venom from B. melanosticus was fractionated by preparative HPLC, and the structures of five new buadienolide sulfates (1-5) along with one new bufogenin (6) were rapidly elucidated on the basis of LC-DAD-MS(n) and LC-SPE-NMR data. The in vitro growth inhibitory activity of these six compounds along with hellebrin (positive control) has been assayed by means of the MTT colorimetric assay in four human and two mouse cancer cell lines. Compound 3 and hellebrin displayed similar and marked in vitro cytotoxicity.

Six new bufadienolides (1-6) and two new C(23) steroids (7 and 8), together with three known compounds (9-11) were isolated from the venom of Bufo bufo gargarizans. Their structures were elucidated by spectroscopic analysis and single-crystal X-ray diffraction. In vitro cytotoxicities of all compounds were evaluated in A549 cancer cell line. Compounds 2, 3 and 10 showed significant cytotoxic activities.

Bufadienolides-loaded nanostructured lipid carriers (BU-NLC) were prepared for parenteral application using glyceryl monostearate as solid core, medium-chain triglyceride and oleic acid as liquid lipid material, and Lipoid E-80, sodium deoxycholate and pluronic F68 as stabilizers. In this study, the in vitro cytotoxicity, pharmacokinetics, biodistribution, antitumor efficacy and safety of BU-NLC were evaluated. Against human astrocytoma cell line (U87-MG) and human gastric carcinoma cell line (HGC-27) BU-NLC exhibited cytotoxicity that was similar to that of the free drug, and superior to that of the commercially available fluorouracil injection. BU-NLC exhibited a linear pharmacokinetic behavior at doses ranging from 0.25 to 1.0 mg/kg. The improved pharmacokinetic profile of bufadienolides when formulated in BU-NLC resulted in a higher plasma concentration and lower clearance after intravenous administration compared with bufadienolides solution (BU-S). A biodistribution study indicated that bufadienolides were mainly distributed in the lung, spleen, brain and kidney, and the longest retention was observed in the brain. A sarcoma-180 tumor model further confirmed the advantages of BU-NLC versus BU-S. Hemolysis and acute toxicity investigations showed that BU-NLC was safe when given by intravenous injection with reduced toxicity. In conclusion, the NLC system is a promising approach for the intravenous delivery of bufadienolides.