OBJECTIVE

To establish the quality standards of Linmaoxiang Jiedu pills.

METHODS

Cinobufagin and bufogenin were determined by HPLC simultaneously.

RESULTS

The average recoverys of Cinobufagin and bufogenin was 100.5% and 100.3%, their linear range were 48.74-731.10 ng and 49.90-748.50 ng, respectively.

CONCLUSIONS

The method for quantification is reproducible and realizable. The method can be used to control quality of Linmaoxiang Jiedu pills as the quality standards.

Toad venom (Chansu) is prepared from the dried secretion of parotid gland and skin gland from Bufo bufo gargarizans or B. melanostictus. Up to now, much attention shall be paid to the poor quality of commercial toad venom because of the adulteration. So, it is urgent to establish a scientific and perfect quality control method to improve the quality of toad venom and guarantee its safety and effectiveness in clinical application. The different batches of toad venom samples were assayed by high performance liquid chromatography (HPLC) and the quantitative analysis of multi-components by single marker (QAMS) was used to detect the contents of five bufagenins. As a result, the reference characteristic chromatogram was established, displaying serotonin, gamabufotalin, arenobufagin, hellebrigenin, telocinobufagin, bufotalin, cinobufotalin, bufalin, cinobufagin and resibufogenin as characteristic peaks. Taking cinobufagin as an internal reference substance, QAMS was verified for the determination of five bufagenins (gamabufotalin, bufotalin, bufalin, cinobufagin, resibufogenin) in toad venom samples. The durability and applicability of the relative correction factor (RCF) were also studied systematically. RCFs of cinobufagin to gamabufotalin, bufotalin, bufalin and resibufogenin were determined as 1.05, 0.895, 1.09 and 0.913, respectively. The characteristic chromatogram and QAMS established in this study could effectively control the quality of toad venom and provide scientific evidence for the improvement of the quality standard of the toad venom to be described in Chinese Pharmacopoeia (2020 edition).

The secreted poisonin bufonids (Anura: Bufonidae) include proteins, biogenic amines, toxic bufadienolides and alkaloids. The chemical composition of the methanolic extract of parotoid gland secretions by the Amazonian toad Rhinella margaritifera was evaluated in a UFLC-DAD-micrOTOF system. Of the twenty three compounds found in the methanolic extract, eighteen were identified by the mass/charge ratio as: five arginine diacids, six bufagenins (telocinobufagin, marinobufagin, bufotalin, cinobufotalin, bufalin and cinobufagin), six bufotoxins, and an alkaloid (dehydrobufotenin).

Objective: To evaluate the analgesic effects of cinobufagin (CBG) on cancer-induced bone pain in rat and study the role of the muscarinic receptor M4 subtype (M4 mAChR) in its involvement. Methods: A total of 100 Female Sprague-Dawley rats were randomly divided into 5 groups (n=Results: At each time point from T(1) to T(6), the mechanical pain thresholds of group S were (8.69±0.45), (8.63±0.44), (8.65±0.39), (8.84±0.23), (8.80±0.14), (8.75±0.14) g, respectively, and the mechanical pain thresholds of group A were (6.37±0.30), (6.42±0.13), (6.29±0.17), (6.25±0.22), (6.34±0.33), (6.36±0.34) g, the difference was statistically significant (t=-16.41, -23.47, -30.25, -17.35, -19.52, -22.56, all P<t=P<t=-12.69, -11.26, -10.33, all P<F=P<F=P>Conclusions: These results demonstrated that M4mAChR participated in mediating the alleviation of hyperalgesia by cinobufagin in rats with bone cancer pain, and its mechanism may be related to pCaMKⅡa/CaMKⅡa signaling pathway.

Traditional Chinese medicine (TCM) has been used for prevention and treatment of various diseases for many decades. TCM injection is a new dosage form, with incidence of anaphylactoid reactions increasing every year. In this study, the rat basophilic leukemia 2H3 (RBL-2H3) and laboratory of allergic disease 2 (LAD2) dual-mixed/CMC was established and was coupled with an HPLC-ESI-IT-TOF-MS system to identify the potential allergenic components in Haqing injection. Cinobufagin, piperine, osthole, praeruptorin A, and schizandrin A were screened from Haqing injection via this coupled system. Competitive binding assay showed piperine, praeruptorin A, and schizandrin A acting on MrgprX2 and cinobufagin and osthole act on the IgE receptor. The release of mediators of anaphylaxis results showed cinobufagin and osthole can cause anaphylactoid reactions by triggering the release of β-hexosaminidase and histamine via IgE-R. Praeruptorin A and schizandrin A could promote the release of β-hexosaminidase and histamine via MrgprX2 receptor. In summary, the dual-mixed/CMC model can significantly improve the efficiency of target component identification from a complex sample. When combined with competitive binding assay and validation of biological activities, this model enables accurate determination of the dual-target components, offering improved methods for quality control of TCM injections.

General pharmacological properties of cinobufagin (CB), isolated from Senso, were compared with those of digitoxin (DT). Decrement of spontaneous movement, inhibition of writhing, prolongation of hexobarbital-induced hypnosis, muscle relaxation, inhibition of acetic acid-induced capillary permeability, hypothermia, antipyretic effect in mice; excitation of respiration in rabbits; nerve blocking action in the isolated sciatic nerve of frogs; cardiotonic effect in the isolated atria of guinea pigs; contraction of the isolated ileum of rabbits and guinea pigs; contraction of the aorta of guinea pigs; and relaxation of the isolated trachea of guinea pigs were common properties observed after separate application of CB and DT. Membrane stabilizing effect in erythrocytes of rats and inhibition of propulsive motility of the small intestine in mice were observed only after the application of CB. Inhibition of gastric juice secretion, antiinflammation on carrageenin- or dextran-induced edema, diuresis in rats, mydriasis in mice and potentiation of transmission in the neuromuscular junction of rats were observed only after the application of DT. After oral administration, the onset of the effects of CB (30 min) was faster than that of DT (3-4 hr), and the duration of the effects of CB (1-2 hr) was shorter than that of DT (greater than 24 hr).

OBJECTIVE

To study the effect of cinobufagin (CB) on the proliferation inhibition and induction of apoptosis in glioblastoma cell lines U87 and its molecular mechanism.

METHODS

A gradient concentration (0-20 μmol/L) of CB was used to treat the U87 glioma cells for 6 h,12 h,24 h and 48 h,respectively. Cell viabilities were determined by CCK-8 assay to discover the effects of different concentrations of CB on the proliferation of glioma cells. Different concentrations (1-20 μmol/L) of CB were used to treat the U87 glioma cells for 12 h and 24 h,hochest33342 staining assay was used to assess the apoptosis levels. Immunofluorescence staining was used to determine the expression of growth related proteins phospho-protein kinase B(T308)[ p-AKT(T308)] in U87 glioma cells after being treated with CB for 24 h. Western blot was used to determine the apoptotic related proteins (BAX,cleaved-caspase 3,cleaved-caspase 9) and growth related proteins [phospho-inositide 3-kinase (p-PI3K),p-AKT(T308),p-AKT(S473),phospho-ribosomal protein S6 kinase (PS6),phospho-4E-binding protein 1 (p-4EBP1)].

RESULTS

A significant effect of CB on the proliferation inhibition and induction of apoptosis in U87 glioma cells in a time- and dose-dependent manner was observed. Treatment with CB induced the expression levels of apoptosis-related protein,cleaved-caspase 3 and BAX,and the PI3K-AKT-4EBP1 signaling pathway related proteins p-AKT(T308) and p-4EBP1 were decreased.

CONCLUSIONS

CB can inhibit U87 glioma cells growth and induce apoptosis,which may involve the PI3K-AKT-4EBP1 and BAX-caspase signaling pathways.

OBJECTIVE

To establish a HPLC method to determine the contents of active constituents in Venenum Bufonis.

METHODS

In determining the contents of resibufogenin and cinobufagin in the traditional Chinese medicine Venenum Bufonis, the mobile phase was acetonitrile-0.5% KH2PO4 solution(50:50)(pH adjusted with value phosphoric acid to 3.25 +/- 0.02).

RESULTS

The constituents thus determined have good linearity and separation. The average recovery of resibufogenin was 100.35%, RSD 1.86%; the average recovery of cinobufagin was 100.38%, RSD 2.09%.

CONCLUSIONS

The method was convenient, rapid, accurate and practicable.

OBJECTIVE

To explore a new experimental method for screening of allergens in post-market traditional Chinese medicine injections by confirming allergens in Huachansu injection.

METHODS

First of all, the serum of patients allergic to Huachansu injection were collected, at the same time, the dubious allergen was conjugated to bovine serum albumin (BSA) by EDC coupling procedure to form complete antigen (BNP-BSA), which makes it possible to reproduce the allergic reaction of Huachansu injection in vitro. The histamine liberation ratio, the level of TNF-alpha and Histamine released from RBL-2H3 mast cell were detected; the above data were compared with those obtained in vivo.

RESULTS

The difference of the histamine liberation ratio, the levels of TNF-alpha and histamine of the resibufogenin-BSA group, group of patients allergic to Huachansu injection were not significant compared with those of normal control group. However, there were significant difference in those data among the cinobufagin-BSA group, the blank control and normal control group (P<0.05).

CONCLUSIONS

The allergen in the serum collected from patients allergic to Huachansu injection is resibufogenin.

OBJECTIVE

To study the chemical constituents of Bufo Siccus.

METHODS

Based on silica column chromatography six compounds were obtained from the alcoholic extract of Bufo Siccus and identified by physico-chemical and spectroscopic analyses.

RESULTS

The compounds were identified as cholesterol, beta-sitosterol, resibufogenin, cinobufagin, bufalin and gamabufotalin.

CONCLUSIONS

Studies on the chemical constituents of Bufo Siccus were reported for the first time.

Actions of cardiac steroid (CS) such as bufadienolides and cardenolides, on guinea pig taenia coli were studied using the double sucrose-gap method. When an appropriate dose of CS was applied, the taenia coli first contracted, then relaxed. After removal of CS, the relaxation was enhanced and continued for 20-40 min. In decreasing order of the relaxant action were bufalin, ouabain, cinobufagin and resibufogenin. Further application of CS after occurrence of the relaxation induced a secondary gradual contraction. The contraction occurred with the membrane depolarization and increase in spike discharge, and the relaxation corresponded fairly well with decrease in spike discharge and the membrane repolarization. Membrane resistance was decreased during the contraction as well as the relaxation. The decrease in membrane resistance continued during the relaxation after removal of CS, Na, K and Ga conductances were increased by CS application, respectively. The increase of Na conductance was relatively high during the contraction, and the increase of K conductance was remarkable during the relaxation and after removal of CS. From the foregoing results it is considered that the contracting action is due to inhibition of the Na pump and that the relaxant action is due to the change in electrical properties of membrane produced by a marked increase in intracellular Na and the increase of K permeability. The relaxation after removal of CS is considered to be due to the activated electrogenic Na pump as indicated from the changes in electrical properties of membrane.

Anoctamin1 (ANO1), a calcium-activated chloride channel, is frequently overexpressed in several cancers, including oral squamous cell carcinoma (OSCC). OSCC is a highly aggressive cancer and the most common oral malignancy. ANO1 has been proposed as a potential candidate for targeted anticancer therapy. In this study, we performed a cell-based screening to identify novel regulators leading to the downregulation of ANO1, and discovered cinobufagin, which downregulated ANO1 expression in oral squamous cell carcinoma CAL-27 cells. ANO1 protein levels were significantly reduced by cinobufagin in a dose-dependent manner with an IC₅₀ value of ~26 nM. Unlike previous ANO1 inhibitors, short-term (≤10 min) exposure to cinobufagin did not alter ANO1 chloride channel activity and ANO1-dependent intestinal smooth muscle contraction, whereas long-term (24 h) exposure to cinobufagin significantly reduced phosphorylation of STAT3 and mRNA expression of ANO1 in CAL-27 cells. Notably, cinobufagin inhibited cell proliferation of CAL-27 cells expressing high levels of ANO1 more potently than that of ANO1 knockout CAL-27 cells. In addition, cinobufagin significantly reduced cell migration and induced caspase-3 activation and PARP cleavage in CAL-27 cells. These results suggest that downregulation of ANO1 by cinobufagin is a potential mechanism for the anticancer effect of cinobufagin in OSCC.

Keywords: CAL-27; STAT3; anoctamin 1; cinobufagin; oral squamous cell carcinoma.

Drying is a critical step to prolong the storage time in natural medicine processing but it changes the chemical characteristics of the product. In this study, research was performed to characterize the metabolomic changes in toad venom induced by vacuum-drying at 60°C and air-drying at room temperature by ultra high performance liquid chromatography coupled with pattern recognition approaches. In total 52 metabolites, down-regulated or up-regulated, were identified as potential chemical markers. Compared with fresh toad venom, vacuum-drying at 60°C succeeded in raising the conjugated-type bufadienolide content significantly, while the content of free-type bufadienolides were slightly reduced. On the other hand, toad venom air-dried at room temperature presented a relatively low amount of bufadienolides compared with fresh venom. For example, the content of several known anti-tumor components (gamabufotalin, bufotalin, cinobufagin, etc.) were significantly reduced. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide bioassay further showed that venom air-dried at room temperature had weaker anti-tumor activity on human hepatocellular carcinoma SMMC-7721 proliferation in vitro than samples vacuum-dried at 60°C. These results showed that the great metabolomic changes of toad venom occurred during the drying process, suggesting that a proper drying procedure is important for sustaining the chemical quality of natural medicines.

Chansu, which is prepared from the skin secretions of toad (Bufo bufo gargarizans Cantor), is widely used in traditional Chinese medicine (TCM). Being the principal bioactive constituents of ChanSu, bufalin (BFL) and cinobufagin (CBF), have been shown to possess anticancer properties. TCM confer bioactivities through the synergistic effect between potential active ingredients, so as to interfere with the development of the disease, and ultimately achieve the therapeutic effect. We found that the anticancer effect was significantly potentiated by co-treatment of BFL and CBF as compared to mono-treatment, suggesting their synergistic interaction. To reveal their synergistic mechanisms, metabolomic and lipidomic profiling based on liquid chromatography-mass spectrometry (LC-MS) were utilized to delineate the responses in HepG2 cells after treatment with BFL and CBF individually or in combination. Metabolic pathways including methionine metabolism, energy metabolism, lipid metabolism and amino acid metabolism were modulated and subsequently lead to apoptosis and cell cycle arrest of HepG2 cells. In particular, the discrepant regulation of methionine metabolism between mono-treatment and co-treatment of BFL and CBF may account for their synergistic effect. Our study provided novel insights into the mechanistic links between cellular metabolism and synergistic effect, which may ultimately lead to better treatments for hepatoma.

Statistical analyses of high-performance liquid chromatography (HPLC)-based chemical fingerprints of Bufo bufo gargarizans toad skin extracts were integrated with antitumor evaluations as a means to more effectively identify bioactive constituents. Specifically, chemical fingerprints of Bufo bufo gargarizans skin extract samples obtained from ten different regions in China were generated by HPLC, and then subjected to similarity analysis and principal component analysis (PCA). Concurrently, a MCF-7 breast cancer cell model was adopted to obtain the antitumor activity of different toad skin extracts. The chromatographic fingerprint-bioactivity relationships of the Bufo bufo gargarizans extracts were then evaluated by Pearson correlation analysis. The results demonstrated that all skin extract samples inhibited the proliferation of MCF-7 cells to some extent, and that there was a correlation between the chemical fingerprints and the anti-proliferative activity. Bufotalin, bufalin, and cinobufagin, three known components of Bufo bufo gargarizans that were identified in the chemical fingerprints, were highlighted as constituents responsible for the observed bioactivity. The activity of cinobufagin was confirmed by cell viability and colony formation assays using MCF-7 cells. The approach documented here provides a more effective means to associate chemical information on medicinal extracts with the principle components responsible for their bioactivity, and to therefore expedite drug discovery.

The pharmacological effects of the toad venom-containing drug "kyushin" on aconitine- and thyroxine-induced arrhythmia in guinea pigs, on the conduction system in Langendorff preparations of rabbit hearts and on the autonomic nervous system in cats were studied. "kyushin" significantly inhibited the aconitine-induced arrhythmia after intraduodenal administration (i.d.) with 80 mg/kg, and the thyroxine-induced arrhythmia with 40 mg/kg i.d. Although "kyushin" itself did not affect the conduction system with 30 mg/ml of the maximal concentration being able to be prepared, bufalin and cinobufagin as constituents of toad venom produced inhibition with 0.3 mg/ml and 1 mg/ml, respectively. The decrease in heart rate induced by electrical stimulation to the parasympathetic nerve (vagus nerve) was potentiated by "kyushin" at 30 mg/kg i.d. The anti-arrhythmic effects of "kyushin" may be attributable to both possible inhibitory effect on the conduction system and potentiating effect on the parasympathetic nervous system.

Uveal melanoma (UM) is the most common primary intraocular carcinoma in adults. Cinobufagin, secreted by the Asiatic toad Bufo gargarizans, is a traditional Chinese medicine, widely used in tumor treatment. Here, we explored the potential antitumor function of cinobufagin and investigated its biochemical mechanisms in UM cells. The antitumor potential of cinobufagin was determined via cell viability, cell cycle, and apoptosis assays. Colony formation assays confirmed that cinobufagin exerted potent antitumor activity in a dose-dependent manner. We found that cinobufagin could induce cell apoptosis and upregulate the expression of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase-9 in vivo and in vitro. In addition, after treatment with increased concentrations of cinobufagin, the intrinsic mitochondrial apoptosis pathway was also activated, which was demonstrated by increased cell apoptosis with increased expression of Bad and Bax, decreased expression of Bcl-2 and Bcl-xl, and reduced mitochondrial membrane potential (MMP) in OCM1 cells. Taken together, the results of this preclinical study suggest that cinobufagin can both inhibit cell survival and induce cell apoptosis in a dose-dependent manner in UM cells, which provides new insights into the biochemical mechanism of cinobufagin and its potential as a future chemotherapeutic agent for UM.

OBJECTIVE

Huqizhengxiao (HQZX) decoction is a mixture of traditional Chinese medicines comprising 10 herbs, with inhibitory effects on hepatocarcinoma. The aim of the study is to observe the antitumor efficacy and mechanism of HQZX decoction in nude mice with hepatocellular carcinoma xenografts.

METHODS

HepG2-luc subcutaneous hepatocarcinoma was established in nude mice. The mice were divided into 5 groups: control, cinobufagin, HQZXS, HQZXM, and HQZXH with doses 13.52, 27.03, and 54.06 g/kg, respectively. HQZX decoction was prepared for intraperitoneal intragastric administration for 3 weeks. Tumor growth was measured with Vernier calipers and in vivo imaging system. α-Fetoprotein (AFP) was determined by radioimmunoassay. Tumor necrosis factor-α (TNF-α) was measured with enzyme-linked immunosorbent assay (ELISA) assay. Telomerase activity was measured with polymerase chain reaction-ELISA. Nuclear mitosis and necrosis were observed with hematoxylin-eosin stain. Apoptotic proteins of caspase-3, Bcl-2, and Bax were examined by Western blot. Signaling molecules of ERK, mTOR, and STAT3 were measured with Luminex assay.

RESULTS

HQZX decoction showed good inhibition of HepG2-luc xenografts. Compared with control group, the relative tumor proliferation rate was less than 60% in the HQZXH and HQZXS. The tumor inhibition rate of HQZXH group reached 52% ± 15%. Relative average optical density values of the HQZXS and HQZXH groups decreased significantly. The mitotic index in HQZXS, HQZXM, and HQZXH groups decreased greatly. Telomerase activity of HQZXS was clearly reduced, and, the caspase-3 expression upregulated in HQZXH group. Bcl-2 expression was downregulated in HQZXS and HQZXH. The ratios of p-ERK/ERK and p-STAT3/STAT3 in HQZXS group were significantly downregulated.

CONCLUSIONS

HQZX decoction can clearly inhibit the growth of hepatocellular carcinoma and induce tumor apoptosis. Its antitumor mechanism may be related to reducing telomerase activity and regulating the STAT3 and ERK signal pathway.

<em>Cinobufagin</em> is a Na<mml:math><mml:msup><mml:mrow></mml:mrow><mml:mrow>+</mml:mrow></mml:msup></mml:math>/K<mml:math><mml:msup><mml:mrow></mml:mrow><mml:mrow>+</mml:mrow></mml:msup></mml:math>-ATPase (NKA) inhibitor with excellent anticancer effects to prolong the survival of patients. The purpose of the present study was to clarify the underlying mechanism of the anticancer effects of <em>cinobufagin</em> using overexpression or inhibition of aurora kinase A (AURKA) signaling. First, high expression of Na<mml:math><mml:msup><mml:mrow></mml:mrow><mml:mrow>+</mml:mrow></mml:msup></mml:math>/K<mml:math><mml:msup><mml:mrow></mml:mrow><mml:mrow>+</mml:mrow></mml:msup></mml:math>-ATPase alpha 1 subunit (ATP1A1) and AURAK resulted in increased malignant transformation in hepatocellular carcinoma (HCC) patients using the cancer genome atlas (TCGA) data and tissue samples. After treatment with <em>cinobufagin</em>, we successfully screened 202, 249, and 335 changing expression proteins in Huh-7 cells under normal, overexpression, and inhibition of AURKA using tandem mass tags (TMT)-labeled quantitative proteomics coupled to 2D liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatics analysis revealed that these molecules were closely associated with chromosome segregation, DNA damage, and regulation of translation processes. We further confirmed that <em>cinobufagin</em> induced DNA damage and chromosome segregation disorders and suppresses translational processing in oncogenes by decreasing the expression of AURKA, mechanistic target of rapamycin kinase (mTOR), p-mTOR, p-extracellular regulated protein kinases (ERK), eukaryotic translation initiation factor 4E (eIF4E), and p-eIF4E, while increasing the expression of p-eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) (S65, T37, T46, T45) and increasing the interaction between eIF4 and 4E-BP1. Our results suggested that <em>cinobufagin</em> performed an antitumor effects in liver cancer cells by inhibiting the AURKA-mTOR-eIF4E axis.

A series of cinobufagin-3-yl nitrogen-containing-carbamate derivatives were designed, synthesized, and evaluated for their proliferation inhibition activities. The structure-activity relationships suggested that the substituents at C-16 was a crucial factor for the potency and that follows this trends: acetic ester ≫ benzoic ester ≈ hydroxy > carbamate. Compounds 3f, 3g, 3h, and 3i exhibited significant in vitro antiproliferative activities against the eight tested tumor cell lines, with IC₅₀ values ranging from 8.1 to 237.4 nM. Furthermore, 3g tartrate (3g-TA) significantly inhibited tumor growth by 64.5%, 83.9%, and 93.0% at a doses of 4, 6, 8 mg/kg/qod by ip, respectively.

Keywords: Antiproliferative activities; Cardiac glycosides; Cinobufagin; Cytotoxicity; Natural product.

Background: Hepatoma is a common malignancy in the world with high morbidity and mortality. The treatment of hepatoma is limited by its poor response to many chemotherapeutic agents. Although paclitaxel (PTX) is widely used in clinical chemotherapy, the low sensitivity to hepatoma restricts its application. Combination therapy is a promising approach to resolve this dilemma.

Objective: To evaluate the interaction between paclitaxel, bufalin (BFL) and cinobufagin (CBF), and explore the optimum combination efficiently.

Methods: HepG2 cells were treated with PTX, BFL and CBF individually or in combination. Their interactions were evaluated by two classical models (Chou-Talalay model and Bliss independence). Response surface methodology (RSM) was used to explore the optimum combination. Furthermore, the optimum drug combination was verified by the morphological experiment.

Results: Synergistic effects were observed when cells were exposed to binary mixtures of PTX+CBF and BFL+CBF. Although the interaction of PTX and BFL was summative, strong synergistic effect was observed when cells were exposed to ternary mixtures of PTX+BFL+CBF. The interaction results of RSM were consistent with classical models, but more efficient. Moreover, the optimum combination dose was given by RSM without the combinatorial explosion of exhaustive testing.

Conclusion: The combination of BFL and CBF synergistically enhanced the potency of PTX against HepG2 cells. RSM could give an accurate evaluation for drug interactions and efficient prediction of optimum combination.

Keywords: Hepatoma; bufalin; cinobufagin; combination treatment; paclitaxel; synergy..

Kinetic properties and crystal structures of the Na⁺,K⁺-ATPase in complex with cardiotonic steroids (CTS) revealed significant differences between CTS subfamilies (Laursen et al.). Thus, we found beneficial effects of K⁺ on bufadienolide binding, which strongly contrasted with the well-known antagonism between K⁺ and cardenolides. In order to understand this peculiarity of bufalin interactions, we used docking and molecular dynamics simulations of the complexes involving Na⁺,K⁺-ATPase, bufadienolides (bufalin, cinobufagin), and ions (K⁺, Na⁺, Mg²⁺). The results revealed that bufadienolide binding is affected by (i) electrostatic attraction of the lactone ring by a cation and (ii) the ability of a cation to stabilize and "shape" the site constituted by transmembrane helices of the α-subunit (αM1-6). The latter effect was due to varying coordination patterns involving amino acid residues from helix bundles αM1-4 and αM5-10. Substituents on the steroid core of a bufadienolide add to and modify the cation effects. The above rationale is fully consistent with the ion effects on the kinetics of Na⁺,K⁺-ATPase/bufadienolide interactions.